Compositions and methods for the therapy and diagnosis of prostate cancer

ABSTRACT

Compositions and methods for the therapy and diagnosis of cancer, particularly prostate cancer, are disclosed. Illustrative compositions comprise one or more prostate-specific polypeptides, immunogenic portions thereof, polynucleotides that encode such polypeptides, antigen presenting cell that expresses such polypeptides, and T cells that are specific for cells expressing such polypeptides. The disclosed compositions are useful, for example, in the diagnosis, prevention and/or treatment of diseases, particularly prostate cancer.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 210121_(—)427C35_SEQUENCE_LISTING.txt. The textfile is 992 KB, was created on Oct. 30, 2007, and is being submittedelectronically via EFS-Web, concurrent with the filing of thespecification.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to therapy and diagnosis ofcancer, such as prostate cancer. The invention is more specificallyrelated to polypeptides, comprising at least a portion of aprostate-specific protein, and to polynucleotides encoding suchpolypeptides. Such polypeptides and polynucleotides are useful inpharmaceutical compositions, e.g., vaccines, and other compositions forthe diagnosis and treatment of prostate cancer.

2. Description of the Related Art

Cancer is a significant health problem throughout the world. Althoughadvances have been made in detection and therapy of cancer, no vaccineor other universally successful method for prevention or treatment iscurrently available. Current therapies, which are generally based on acombination of chemotherapy or surgery and radiation, continue to proveinadequate in many patients.

Prostate cancer is the most common form of cancer among males, with anestimated incidence of 30% in men over the age of 50. Overwhelmingclinical evidence shows that human prostate cancer has the propensity tometastasize to bone, and the disease appears to progress inevitably fromandrogen dependent to androgen refractory status, leading to increasedpatient mortality. This prevalent disease is currently the secondleading cause of cancer death among men in the U.S.

In spite of considerable research into therapies for the disease,prostate cancer remains difficult to treat. Commonly, treatment is basedon surgery and/or radiation therapy, but these methods are ineffectivein a significant percentage of cases. Two previously identified prostatespecific proteins—prostate specific antigen (PSA) and prostatic acidphosphatase (PAP)—have limited therapeutic and diagnostic potential. Forexample, PSA levels do not always correlate well with the presence ofprostate cancer, being positive in a percentage of non-prostate cancercases, including benign prostatic hyperplasia (BPH). Furthermore, PSAmeasurements correlate with prostate volume, and do not indicate thelevel of metastasis.

In spite of considerable research into therapies for these and othercancers, prostate cancer remains difficult to diagnose and treateffectively. Accordingly, there is a need in the art for improvedmethods for detecting and treating such cancers. The present inventionfulfills these needs and further provides other related advantages.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides polynucleotidecompositions comprising a sequence selected from the group consistingof:

(a) sequences provided in SEQ ID NO: 1-111, 115-171, 173-175, 177,179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476,524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606,618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907,908, 916-919, 929-931, 938, 939, 942, 944, 945, 948, 967, 969-976, 991,993-1002, 1004, 1006, 1007, 1010, 1013-1019, 1021, 1023-1027, and1034-1036;

(b) complements of the sequences provided in SEQ ID NO: 1-111, 115-171,173-175, 177,179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591,593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894,896, 907, 908, 916-919, 929-931, 938, 939, 942, 944, 945, 948, 967,969-976, 991, 993-1002, 1004, 1006, 1007, 1010, 1013-1019, 1021,1023-1027, and 1034-1036;

(c) sequences consisting of at least 20 contiguous residues of asequence provided in SEQ ID NO: 1-111, 115-171, 173-175, 177,179-305,307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524,526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705,709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908,916-919, 929-931, 938, 939, 942, 944, 945, 948, 967, 969-976, 991,993-1002, 1004, 1006, 1007, 1010, 1013-1019, 1021, 1023-1027, and1034-1036;

(d) sequences that hybridize to a sequence provided in SEQ ID NO: 1-111,115-171, 173-175, 177,179-305, 307-315, 326, 328, 330, 332-335, 340-375,381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572,587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878,880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939, 942, 944, 945,948, 967, 969-976, 991, 993-1002, 1004, 1006, 1007, 1010, 1013-1019,1021, 1023-1027, and 1034-1036, under moderately stringent conditions;

(e) sequences having at least 75% identity to a sequence of SEQ ID NO:1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335,340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552,569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824,878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939, 942, 944,945, 948, 967, 969-976, 991, 993-1002, 1004, 1006, 1007, 1010,1013-1019, 1021, 1023-1027, and 1034-1036;

(f) sequences having at least 90% identity to a sequence of SEQ ID NO:1-111, 115-171, 173-175, 177,179-305, 307-315, 326, 328, 330, 332-335,340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552,569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824,878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939, 942, 944,945, 948, 967, 969-976, 991, 993-1002, 1004, 1006, 1007, 1010,1013-1019, 1021, 1023-1027, and 1034-1036; and

(g) degenerate variants of a sequence provided in SEQ ID NO: 1-111,115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335,340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552,569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824,878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939, 942, 944,945, 948, 967, 969-976, 991, 993-1002, 1004, 1006, 1007, 1010,1013-1019, 1021, 1023-1027, and 1034-1036.

In one preferred embodiment, the polynucleotide compositions of theinvention are expressed in at least about 20%, more preferably in atleast about 30%, and most preferably in at least about 50% of prostatetissue samples tested, at a level that is at least about 2-fold,preferably at least about 5-fold, and most preferably at least about10-fold higher than that for other normal tissues.

The present invention, in another aspect, provides polypeptidecompositions comprising an amino acid sequence that is encoded by apolynucleotide sequence described above.

The present invention further provides polypeptide compositionscomprising an amino acid sequence selected from the group consisting ofsequences recited in SEQ ID NO: 112-114, 172, 176, 178, 327, 329, 331,336, 339, 376-380, 383, 477-483, 496, 504, 505, 519, 520, 522, 525, 527,532, 534, 537-551, 553-568, 573-586, 588-590, 592, 706-708, 775, 776,778, 780, 781, 811, 814, 818, 826, 827, 853, 855, 858, 860-862, 866-877,879, 883-893, 895, 897, 898, 909-915, 920-928, 932-934, 940, 941, 943,946, 947, 949-966, 968, 977-990, 992, 1003, 1005, 1008, 1009, 1011,1012, 1020, 1022, 1028-1029, 1030-1033, and 1037.

In certain preferred embodiments, the polypeptides and/orpolynucleotides of the present invention are immunogenic, i.e., they arecapable of eliciting an immune response, particularly a humoral and/orcellular immune response, as further described herein.

In another illustrative embodiment, a P501S (SEQ ID NO: 113) polypeptideof the present invention comprises a fragment of P501S that willminimally comprise SEQ ID NO:1037, which represents an 11 amino acidfragment of P501S that was unexpectedly found to contain naturallyprocessed epitopes for at least three Class I MHC alleles. In a relatedembodiment, the polypeptides of the present invention comprise fragmentsof P501S that minimally contain SEQ ID NO:1037, and that are at least12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380,381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394,395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408,409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422,423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436,437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450,451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464,465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478,479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492,493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506,507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520,521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534,535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548,549, 550, 551, 552, or 553, amino acids in length and contain SEQ IDNO:1037.

Certain other illustrative P501S polypeptides according to thisembodiment will consist of at least 11-542 amino acid residues, 11-100amino acid residues, 11-50 amino acid residues, or 11-25 amino acidresidues of SEQ ID NO: 113 and will contain SEQ ID NO: 1037.

The present invention further provides fragments, variants and/orderivatives of the disclosed polypeptide and/or polynucleotidesequences, wherein the fragments, variants and/or derivatives preferablyhave a level of immunogenic activity of at least about 50%, preferablyat least about 70% and more preferably at least about 90% of the levelof immunogenic activity of a polypeptide sequence set forth in SEQ IDNO: 112-114, 172, 176, 178, 327, 329, 331, 336, 339, 376-380, 383,477-483, 496, 504, 505, 519, 520, 522, 525, 527, 532, 534, 537-551,553-568, 573-586, 588-590, 592, 706-708, 775, 776, 778, 780, 781, 811,814, 818, 826, 827, 853, 855, 858 or 860-862, 866-877, 879, 883-893,895, 897, 898, 909-915, 920-928, 932-934, 940, 941, 943, 946, 947,949-966, 968, 977-990, 992, 1003, 1005, 1008, 1009, 1011, 1012, 1020,1022, 1028-1029, and 1030-1033 or a polypeptide sequence encoded by apolynucleotide sequence set forth in SEQ ID NO: 1-111, 115-171, 173-175,177, 179-305, 307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and384-476, 524, 526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591,593-606, 618-705, 709-774, 777, 789, 817, 823, 824, 878, 880-882, 894,896, 907, 908, 916-919, 929-931, 938, 939, 942, 944, 945, 948, 967,969-976, 991, 993-1002, 1004, 1006, 1007, 1010, 1013-1019, 1021,1023-1027, and 1034-1037.

The present invention further provides polynucleotides that encode apolypeptide described above, expression vectors comprising suchpolynucleotides and host cells transformed or transfected with suchexpression vectors.

Within other aspects, the present invention provides pharmaceuticalcompositions comprising a polypeptide or polynucleotide as describedabove and a physiologically acceptable carrier.

Within a related aspect of the present invention, pharmaceuticalcompositions, e.g., vaccine compositions, are provided for prophylacticor therapeutic applications. Such compositions generally comprise animmunogenic polypeptide or polynucleotide of the invention and animmunostimulant, such as an adjuvant, together with a physiologicallyacceptable carrier.

The present invention further provides pharmaceutical compositions thatcomprise: (a) an antibody or antigen-binding fragment thereof thatspecifically binds to a polypeptide of the present invention, or afragment thereof; and (b) a physiologically acceptable carrier.

Within further aspects, the present invention provides pharmaceuticalcompositions comprising: (a) an antigen presenting cell that expresses apolypeptide as described above and (b) a pharmaceutically acceptablecarrier or excipient. Illustrative antigen presenting cells includedendritic cells, macrophages, monocytes, fibroblasts and B cells.

Within related aspects, pharmaceutical compositions are provided thatcomprise: (a) an antigen presenting cell that expresses a polypeptide asdescribed above and (b) an immunostimulant.

The present invention further provides, in other aspects, fusionproteins that comprise at least one polypeptide as described above, aswell as polynucleotides encoding such fusion proteins, typically in theform of pharmaceutical compositions, e.g., vaccine compositions,comprising a physiologically acceptable carrier and/or animmunostimulant. The fusions proteins may comprise multiple immunogenicpolypeptides or portions/variants thereof, as described herein, and mayfurther comprise one or more polypeptide segments for facilitatingand/or enhancing the expression, purification and/or immunogenicity ofthe polypeptide(s).

Within further aspects, the present invention provides methods forstimulating an immune response in a patient, preferably a T cellresponse in a human patient, comprising administering a pharmaceuticalcomposition described herein. The patient may be afflicted with prostatecancer, in which case the methods provide treatment for the disease, ora patient considered to be at risk for such a disease may be treatedprophylactically.

Within further aspects, the present invention provides methods forinhibiting the development of a cancer in a patient, comprisingadministering to a patient a pharmaceutical composition as recitedabove. The patient may be afflicted with prostate cancer, in which casethe methods provide treatment for the disease, or a patient consideredto be at risk for such a disease may be treated prophylactically.

The present invention further provides, within other aspects, methodsfor removing tumor cells from a biological sample, comprising contactinga biological sample with T cells that specifically react with apolypeptide of the present invention, wherein the step of contacting isperformed under conditions and for a time sufficient to permit theremoval of cells expressing the polypeptide from the sample.

Within related aspects, methods are provided for inhibiting thedevelopment of a cancer in a patient, comprising administering to apatient a biological sample treated as described above.

Methods are further provided, within other aspects, for stimulatingand/or expanding T cells specific for a polypeptide of the presentinvention, comprising contacting T cells with one or more of: (i) apolypeptide as described above; (ii) a polynucleotide encoding such apolypeptide; and (iii) an antigen presenting cell that expresses such apolypeptide; under conditions and for a time sufficient to permit thestimulation and/or expansion of T cells. Isolated T cell populationscomprising T cells prepared as described above are also provided.

Within further aspects, the present invention provides methods forinhibiting the development of a cancer in a patient, comprisingadministering to a patient an effective amount of a T cell population asdescribed above.

The present invention further provides methods for inhibiting thedevelopment of a cancer in a patient, comprising the steps of: (a)incubating CD4⁺ and/or CD8⁺ T cells isolated from a patient with one ormore of: (i) a polypeptide comprising at least an immunogenic portion ofpolypeptide disclosed herein; (ii) a polynucleotide encoding such apolypeptide; and (iii) an antigen-presenting cell that expressed such apolypeptide; and (b) administering to the patient an effective amount ofthe proliferated T cells, thereby inhibiting the development of a cancerin the patient. Proliferated cells may, but need not, be cloned prior toadministration to the patient.

Within further aspects, the present invention provides methods fordetermining the presence or absence of a cancer, preferably a prostatecancer, in a patient comprising: (a) contacting a biological sampleobtained from a patient with a binding agent that binds to a polypeptideas recited above; (b) detecting in the sample an amount of polypeptidethat binds to the binding agent; and (c) comparing the amount ofpolypeptide with a predetermined cut-off value, and therefromdetermining the presence or absence of a cancer in the patient. Withinpreferred embodiments, the binding agent is an antibody, more preferablya monoclonal antibody.

The present invention also provides, within other aspects, methods formonitoring the progression of a cancer in a patient. Such methodscomprise the steps of: (a) contacting a biological sample obtained froma patient at a first point in time with a binding agent that binds to apolypeptide as recited above; (b) detecting in the sample an amount ofpolypeptide that binds to the binding agent; (c) repeating steps (a) and(b) using a biological sample obtained from the patient at a subsequentpoint in time; and (d) comparing the amount of polypeptide detected instep (c) with the amount detected in step (b), and therefrom monitoringthe progression of the cancer in the patient.

The present invention further provides, within other aspects, methodsfor determining the presence or absence of a cancer in a patient,comprising the steps of: (a) contacting a biological sample obtainedfrom a patient with an oligonucleotide that hybridizes to apolynucleotide of the present invention; (b) detecting in the sample alevel of a polynucleotide, preferably mRNA, that hybridizes to theoligonucleotide; and (c) comparing the level of polynucleotide thathybridizes to the oligonucleotide with a predetermined cut-off value,and therefrom determining the presence or absence of a cancer in thepatient. Within certain embodiments, the amount of mRNA is detected viapolymerase chain reaction using, for example, at least oneoligonucleotide primer that hybridizes to a polynucleotide of thepresent invention, or a complement of such a polynucleotide. Withinother embodiments, the amount of mRNA is detected using a hybridizationtechnique, employing an oligonucleotide probe that hybridizes to aninventive polynucleotide, or a complement of such a polynucleotide.

In related aspects, methods are provided for monitoring the progressionof a cancer in a patient, comprising the steps of: (a) contacting abiological sample obtained from a patient with an oligonucleotide thathybridizes to a polynucleotide of the present invention; (b) detectingin the sample an amount of a polynucleotide that hybridizes to theoligonucleotide; (c) repeating steps (a) and (b) using a biologicalsample obtained from the patient at a subsequent point in time; and (d)comparing the amount of polynucleotide detected in step (c) with theamount detected in step (b), and therefrom monitoring the progression ofthe cancer in the patient.

Within further aspects, the present invention provides antibodies, suchas monoclonal antibodies, that bind to a polypeptide as described above,as well as diagnostic kits comprising such antibodies. Diagnostic kitscomprising one or more oligonucleotide probes or primers as describedabove are also provided.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE IDENTIFIERS

FIG. 1 illustrates the ability of T cells to kill fibroblasts expressingthe representative prostate-specific polypeptide P502S, as compared tocontrol fibroblasts. The percentage lysis is shown as a series ofeffector:target ratios, as indicated.

FIGS. 2A and 2B illustrate the ability of T cells to recognize cellsexpressing the representative prostate-specific polypeptide P502S. Ineach case, the number of γ-interferon spots is shown for differentnumbers of responders. In FIG. 2A, data is presented for fibroblastspulsed with the P2S-12 peptide, as compared to fibroblasts pulsed with acontrol E75 peptide. In FIG. 2B, data is presented for fibroblastsexpressing P502S, as compared to fibroblasts expressing HER-2/neu.

FIG. 3 represents a peptide competition binding assay showing that theP1S#10 peptide, derived from P501S, binds HLA-A2. Peptide P1S#10inhibits HLA-A2 restricted presentation of fluM58 peptide to CTL cloneD150M58 in TNF release bioassay. D150M58 CTL is specific for the HLA-A2binding influenza matrix peptide fluM58.

FIG. 4 illustrates the ability of T cell lines generated from P1S#10immunized mice to specifically lyse P1S#10-pulsed Jurkat A2Kb targetsand P501S-transduced Jurkat A2Kb targets, as compared to EGFP-transducedJurkat A2Kb. The percent lysis is shown as a series of effector totarget ratios, as indicated.

FIG. 5 illustrates the ability of a T cell clone to recognize andspecifically lyse Jurkat A2Kb cells expressing the representativeprostate-specific polypeptide P501S, thereby demonstrating that theP1S#10 peptide may be a naturally processed epitope of the P501Spolypeptide.

FIGS. 6A and 6B are graphs illustrating the specificity of a CD8⁺ cellline (3A-1) for a representative prostate-specific antigen (P501S). FIG.6A shows the results of a ⁵¹Cr release assay. The percent specific lysisis shown as a series of effector:target ratios, as indicated. FIG. 6Bshows the production of interferon-gamma by 3A-1 cells stimulated withautologous B-LCL transduced with P501S, at varying effector:targetrations as indicated.

FIG. 7 is a Western blot showing the expression of P501S in baculovirus.

FIG. 8 illustrates the results of epitope mapping studies on P501S.

FIG. 9 is a schematic representation of the P501S protein showing thelocation of transmembrane domains and predicted intracellular andextracellular domains.

FIG. 10 is a genomic map showing the location of the prostate genesP775P, P704P, B305D, P712P and P774P within the Cat Eye Syndrome regionof chromosome 22q11.2

FIG. 11 shows the results of an ELISA assay to determine the specificityof rabbit polyclonal antisera raised against P501S.

SEQ ID NO: 1 is the determined cDNA sequence for F1-13

SEQ ID NO: 2 is the determined 3′ cDNA sequence for F1-12

SEQ ID NO: 3 is the determined 5′ cDNA sequence for F1-12

SEQ ID NO: 4 is the determined 3′ cDNA sequence for F1-16

SEQ ID NO: 5 is the determined 3′ cDNA sequence for H1-1

SEQ ID NO: 6 is the determined 3′ cDNA sequence for H1-9

SEQ ID NO: 7 is the determined 3′ cDNA sequence for H1-4

SEQ ID NO: 8 is the determined 3′ cDNA sequence for J1-17

SEQ ID NO: 9 is the determined 5′ cDNA sequence for J1-17

SEQ ID NO: 10 is the determined 3′ cDNA sequence for L1-12

SEQ ID NO: 11 is the determined 5′ cDNA sequence for L1-12

SEQ ID NO: 12 is the determined 3′ cDNA sequence for N1-1862

SEQ ID NO: 13 is the determined 5′ cDNA sequence for N1-1862

SEQ ID NO: 14 is the determined 3′ cDNA sequence for J1-13

SEQ ID NO: 15 is the determined 5′ cDNA sequence for J1-13

SEQ ID NO: 16 is the determined 3′ cDNA sequence for J1-19

SEQ ID NO: 17 is the determined 5′ cDNA sequence for J1-19

SEQ ID NO: 18 is the determined 3′ cDNA sequence for J1-25

SEQ ID NO: 19 is the determined 5′ cDNA sequence for J1-25

SEQ ID NO: 20 is the determined 5′ cDNA sequence for J1-24

SEQ ID NO: 21 is the determined 3′ cDNA sequence for J1-24

SEQ ID NO: 22 is the determined 5′ cDNA sequence for K1-58

SEQ ID NO: 23 is the determined 3′ cDNA sequence for K1-58

SEQ ID NO: 24 is the determined 5′ cDNA sequence for K1-63

SEQ ID NO: 25 is the determined 3′ cDNA sequence for K1-63

SEQ ID NO: 26 is the determined 5′ cDNA sequence for L1-4

SEQ ID NO: 27 is the determined 3′ cDNA sequence for L1-4

SEQ ID NO: 28 is the determined 5′ cDNA sequence for L1-14

SEQ ID NO: 29 is the determined 3′ cDNA sequence for L1-14

SEQ ID NO: 30 is the determined 3′ cDNA sequence for J1-12

SEQ ID NO: 31 is the determined 3′ cDNA sequence for J1-16

SEQ ID NO: 32 is the determined 3′ cDNA sequence for J1-21

SEQ ID NO: 33 is the determined 3′ cDNA sequence for K1-48

SEQ ID NO: 34 is the determined 3′ cDNA sequence for K1-55

SEQ ID NO: 35 is the determined 3′ cDNA sequence for L1-2

SEQ ID NO: 36 is the determined 3′ cDNA sequence for L1-6

SEQ ID NO: 37 is the determined 3′ cDNA sequence for N1-1858

SEQ ID NO: 38 is the determined 3′ cDNA sequence for N1-1860

SEQ ID NO: 39 is the determined 3′ cDNA sequence for N1-1861

SEQ ID NO: 40 is the determined 3′ cDNA sequence for N1-1864

SEQ ID NO: 41 is the determined cDNA sequence for P5

SEQ ID NO: 42 is the determined cDNA sequence for P8

SEQ ID NO: 43 is the determined cDNA sequence for P9

SEQ ID NO: 44 is the determined cDNA sequence for P18

SEQ ID NO: 45 is the determined cDNA sequence for P20

SEQ ID NO: 46 is the determined cDNA sequence for P29

SEQ ID NO: 47 is the determined cDNA sequence for P30

SEQ ID NO: 48 is the determined cDNA sequence for P34

SEQ ID NO: 49 is the determined cDNA sequence for P36

SEQ ID NO: 50 is the determined cDNA sequence for P38

SEQ ID NO: 51 is the determined cDNA sequence for P39

SEQ ID NO: 52 is the determined cDNA sequence for P42

SEQ ID NO: 53 is the determined cDNA sequence for P47

SEQ ID NO: 54 is the determined cDNA sequence for P49

SEQ ID NO: 55 is the determined cDNA sequence for P50

SEQ ID NO: 56 is the determined cDNA sequence for P53

SEQ ID NO: 57 is the determined cDNA sequence for P55

SEQ ID NO: 58 is the determined cDNA sequence for P60

SEQ ID NO: 59 is the determined cDNA sequence for P64

SEQ ID NO: 60 is the determined cDNA sequence for P65

SEQ ID NO: 61 is the determined cDNA sequence for P73

SEQ ID NO: 62 is the determined cDNA sequence for P75

SEQ ID NO: 63 is the determined cDNA sequence for P76

SEQ ID NO: 64 is the determined cDNA sequence for P79

SEQ ID NO: 65 is the determined cDNA sequence for P84

SEQ ID NO: 66 is the determined cDNA sequence for P68

SEQ ID NO: 67 is the determined cDNA sequence for P80 (also referred toas P704P)

SEQ ID NO: 68 is the determined cDNA sequence for P82

SEQ ID NO: 69 is the determined cDNA sequence for U1-3064

SEQ ID NO: 70 is the determined cDNA sequence for U1-3065

SEQ ID NO: 71 is the determined cDNA sequence for V1-3692

SEQ ID NO: 72 is the determined cDNA sequence for 1A-3905

SEQ ID NO: 73 is the determined cDNA sequence for V1-3686

SEQ ID NO: 74 is the determined cDNA sequence for R1-2330

SEQ ID NO: 75 is the determined cDNA sequence for 1B-3976

SEQ ID NO: 76 is the determined cDNA sequence for V1-3679

SEQ ID NO: 77 is the determined cDNA sequence for 1G-4736

SEQ ID NO: 78 is the determined cDNA sequence for 1G-4738

SEQ ID NO: 79 is the determined cDNA sequence for 1G-4741

SEQ ID NO: 80 is the determined cDNA sequence for 1G-4744

SEQ ID NO: 81 is the determined cDNA sequence for 1G-4734

SEQ ID NO: 82 is the determined cDNA sequence for 1H-4774

SEQ ID NO: 83 is the determined cDNA sequence for 1H-4781

SEQ ID NO: 84 is the determined cDNA sequence for 1H-4785

SEQ ID NO: 85 is the determined cDNA sequence for 1H-4787

SEQ ID NO: 86 is the determined cDNA sequence for 1H-4796

SEQ ID NO: 87 is the determined cDNA sequence for 1I-4807

SEQ ID NO: 88 is the determined cDNA sequence for 1I-4810

SEQ ID NO: 89 is the determined cDNA sequence for 1I-4811

SEQ ID NO: 90 is the determined cDNA sequence for 1J-4876

SEQ ID NO: 91 is the determined cDNA sequence for 1K-4884

SEQ ID NO: 92 is the determined cDNA sequence for 1K-4896

SEQ ID NO: 93 is the determined cDNA sequence for 1G-4761

SEQ ID NO: 94 is the determined cDNA sequence for 1G-4762

SEQ ID NO: 95 is the determined cDNA sequence for 1H-4766

SEQ ID NO: 96 is the determined cDNA sequence for 1H-4770

SEQ ID NO: 97 is the determined cDNA sequence for 1H-4771

SEQ ID NO: 98 is the determined cDNA sequence for 1H-4772

SEQ ID NO: 99 is the determined cDNA sequence for 1D-4297

SEQ ID NO: 100 is the determined cDNA sequence for 1D-4309

SEQ ID NO: 101 is the determined cDNA sequence for 1D.1-4278

SEQ ID NO: 102 is the determined cDNA sequence for 1D-4288

SEQ ID NO: 103 is the determined cDNA sequence for 1D-4283

SEQ ID NO: 104 is the determined cDNA sequence for 1D-4304

SEQ ID NO: 105 is the determined cDNA sequence for 1D-4296

SEQ ID NO: 106 is the determined cDNA sequence for 1D-4280

SEQ ID NO: 107 is the determined full length cDNA sequence for F1-12(also referred to as P504S)

SEQ ID NO: 108 is the amino acid sequence for F1-12

SEQ ID NO: 109 is the determined full length cDNA sequence for J1-17

SEQ ID NO: 110 is the determined full length cDNA sequence for L1-12(also referred to as P501S)

SEQ ID NO: 111 is the determined full length cDNA sequence for N1-1862(also referred to as P503S)

SEQ ID NO: 112 is the amino acid sequence for J1-17

SEQ ID NO: 113 is the amino acid sequence for L1-12 (also referred to asP501S)

SEQ ID NO: 114 is the amino acid sequence for N1-1862 (also referred toas P503S)

SEQ ID NO: 115 is the determined cDNA sequence for P89

SEQ ID NO: 116 is the determined cDNA sequence for P90

SEQ ID NO: 117 is the determined cDNA sequence for P92

SEQ ID NO: 118 is the determined cDNA sequence for P95

SEQ ID NO: 119 is the determined cDNA sequence for P98

SEQ ID NO: 120 is the determined cDNA sequence for P102

SEQ ID NO: 121 is the determined cDNA sequence for P110

SEQ ID NO: 122 is the determined cDNA sequence for P111

SEQ ID NO: 123 is the determined cDNA sequence for P114

SEQ ID NO: 124 is the determined cDNA sequence for P115

SEQ ID NO: 125 is the determined cDNA sequence for P116

SEQ ID NO: 126 is the determined cDNA sequence for P124

SEQ ID NO: 127 is the determined cDNA sequence for P126

SEQ ID NO: 128 is the determined cDNA sequence for P130

SEQ ID NO: 129 is the determined cDNA sequence for P133

SEQ ID NO: 130 is the determined cDNA sequence for P138

SEQ ID NO: 131 is the determined cDNA sequence for P143

SEQ ID NO: 132 is the determined cDNA sequence for P151

SEQ ID NO: 133 is the determined cDNA sequence for P156

SEQ ID NO: 134 is the determined cDNA sequence for P157

SEQ ID NO: 135 is the determined cDNA sequence for P166

SEQ ID NO: 136 is the determined cDNA sequence for P176

SEQ ID NO: 137 is the determined cDNA sequence for P178

SEQ ID NO: 138 is the determined cDNA sequence for P179

SEQ ID NO: 139 is the determined cDNA sequence for P185

SEQ ID NO: 140 is the determined cDNA sequence for P192

SEQ ID NO: 141 is the determined cDNA sequence for P201

SEQ ID NO: 142 is the determined cDNA sequence for P204

SEQ ID NO: 143 is the determined cDNA sequence for P208

SEQ ID NO: 144 is the determined cDNA sequence for P211

SEQ ID NO: 145 is the determined cDNA sequence for P213

SEQ ID NO: 146 is the determined cDNA sequence for P219

SEQ ID NO: 147 is the determined cDNA sequence for P237

SEQ ID NO: 148 is the determined cDNA sequence for P239

SEQ ID NO: 149 is the determined cDNA sequence for P248

SEQ ID NO: 150 is the determined cDNA sequence for P251

SEQ ID NO: 151 is the determined cDNA sequence for P255

SEQ ID NO: 152 is the determined cDNA sequence for P256

SEQ ID NO: 153 is the determined cDNA sequence for P259

SEQ ID NO: 154 is the determined cDNA sequence for P260

SEQ ID NO: 155 is the determined cDNA sequence for P263

SEQ ID NO: 156 is the determined cDNA sequence for P264

SEQ ID NO: 157 is the determined cDNA sequence for P266

SEQ ID NO: 158 is the determined cDNA sequence for P270

SEQ ID NO: 159 is the determined cDNA sequence for P272

SEQ ID NO: 160 is the determined cDNA sequence for P278

SEQ ID NO: 161 is the determined cDNA sequence for P105

SEQ ID NO: 162 is the determined cDNA sequence for P107

SEQ ID NO: 163 is the determined cDNA sequence for P137

SEQ ID NO: 164 is the determined cDNA sequence for P194

SEQ ID NO: 165 is the determined cDNA sequence for P195

SEQ ID NO: 166 is the determined cDNA sequence for P196

SEQ ID NO: 167 is the determined cDNA sequence for P220

SEQ ID NO: 168 is the determined cDNA sequence for P234

SEQ ID NO: 169 is the determined cDNA sequence for P235

SEQ ID NO: 170 is the determined cDNA sequence for P243

SEQ ID NO: 171 is the determined cDNA sequence for P703P-DE1

SEQ ID NO: 172 is the amino acid sequence for P703P-DE1

SEQ ID NO: 173 is the determined cDNA sequence for P703P-DE2

SEQ ID NO: 174 is the determined cDNA sequence for P703P-DE6

SEQ ID NO: 175 is the determined cDNA sequence for P703P-DE13

SEQ ID NO: 176 is the amino acid sequence for P703P-DE13

SEQ ID NO: 177 is the determined cDNA sequence for P703P-DE14

SEQ ID NO: 178 is the amino acid sequence for P703P-DE14

SEQ ID NO: 179 is the determined extended cDNA sequence for 1G-4736

SEQ ID NO: 180 is the determined extended cDNA sequence for 1G-4738

SEQ ID NO: 181 is the determined extended cDNA sequence for 1G-4741

SEQ ID NO: 182 is the determined extended cDNA sequence for 1G-4744

SEQ ID NO: 183 is the determined extended cDNA sequence for 1H-4774

SEQ ID NO: 184 is the determined extended cDNA sequence for 1H-4781

SEQ ID NO: 185 is the determined extended cDNA sequence for 1H-4785

SEQ ID NO: 186 is the determined extended cDNA sequence for 1H-4787

SEQ ID NO: 187 is the determined extended cDNA sequence for 1H-4796

SEQ ID NO: 188 is the determined extended cDNA sequence for 1I-4807

SEQ ID NO: 189 is the determined 3′ cDNA sequence for 1I-4810

SEQ ID NO: 190 is the determined 3′ cDNA sequence for 1I-4811

SEQ ID NO: 191 is the determined extended cDNA sequence for 1I-4876

SEQ ID NO: 192 is the determined extended cDNA sequence for 1K-4884

SEQ ID NO: 193 is the determined extended cDNA sequence for 1K-4896

SEQ ID NO: 194 is the determined extended cDNA sequence for 1G-4761

SEQ ID NO: 195 is the determined extended cDNA sequence for 1G-4762

SEQ ID NO: 196 is the determined extended cDNA sequence for 1H-4766

SEQ ID NO: 197 is the determined 3′ cDNA sequence for 1H-4770

SEQ ID NO: 198 is the determined 3′ cDNA sequence for 1H-4771

SEQ ID NO: 199 is the determined extended cDNA sequence for 1H-4772

SEQ ID NO: 200 is the determined extended cDNA sequence for 1D-4309

SEQ ID NO: 201 is the determined extended cDNA sequence for 1D.1-4278

SEQ ID NO: 202 is the determined extended cDNA sequence for 1D-4288

SEQ ID NO: 203 is the determined extended cDNA sequence for 1D-4283

SEQ ID NO: 204 is the determined extended cDNA sequence for 1D-4304

SEQ ID NO: 205 is the determined extended cDNA sequence for 1D-4296

SEQ ID NO: 206 is the determined extended cDNA sequence for 1D-4280

SEQ ID NO: 207 is the determined cDNA sequence for 10-d8fwd

SEQ ID NO: 208 is the determined cDNA sequence for 10-H10con

SEQ ID NO: 209 is the determined cDNA sequence for 11-C8rev

SEQ ID NO: 210 is the determined cDNA sequence for 7.g6fwd

SEQ ID NO: 211 is the determined cDNA sequence for 7.g6rev

SEQ ID NO: 212 is the determined cDNA sequence for 8-b5fwd

SEQ ID NO: 213 is the determined cDNA sequence for 8-b5rev

SEQ ID NO: 214 is the determined cDNA sequence for 8-b6fwd

SEQ ID NO: 215 is the determined cDNA sequence for 8-b6 rev

SEQ ID NO: 216 is the determined cDNA sequence for 8-d4fwd

SEQ ID NO: 217 is the determined cDNA sequence for 8-d9rev

SEQ ID NO: 218 is the determined cDNA sequence for 8-g3fwd

SEQ ID NO: 219 is the determined cDNA sequence for 8-g3rev

SEQ ID NO: 220 is the determined cDNA sequence for 8-h11rev

SEQ ID NO: 221 is the determined cDNA sequence for g-f12fwd

SEQ ID NO: 222 is the determined cDNA sequence for g-f3rev

SEQ ID NO: 223 is the determined cDNA sequence for P509S

SEQ ID NO: 224 is the determined cDNA sequence for P510S

SEQ ID NO: 225 is the determined cDNA sequence for P703DE5

SEQ ID NO: 226 is the determined cDNA sequence for 9-A11

SEQ ID NO: 227 is the determined cDNA sequence for 8-C6

SEQ ID NO: 228 is the determined cDNA sequence for 8-H7

SEQ ID NO: 229 is the determined cDNA sequence for JPTPN13

SEQ ID NO: 230 is the determined cDNA sequence for JPTPN14

SEQ ID NO: 231 is the determined cDNA sequence for JPTPN23

SEQ ID NO: 232 is the determined cDNA sequence for JPTPN24

SEQ ID NO: 233 is the determined cDNA sequence for JPTPN25

SEQ ID NO: 234 is the determined cDNA sequence for JPTPN30

SEQ ID NO: 235 is the determined cDNA sequence for JPTPN34

SEQ ID NO: 236 is the determined cDNA sequence for PTPN35

SEQ ID NO: 237 is the determined cDNA sequence for JPTPN36

SEQ ID NO: 238 is the determined cDNA sequence for JPTPN38

SEQ ID NO: 239 is the determined cDNA sequence for JPTPN39

SEQ ID NO: 240 is the determined cDNA sequence for JPTPN40

SEQ ID NO: 241 is the determined cDNA sequence for JPTPN41

SEQ ID NO: 242 is the determined cDNA sequence for JPTPN42

SEQ ID NO: 243 is the determined cDNA sequence for JPTPN45

SEQ ID NO: 244 is the determined cDNA sequence for JPTPN46

SEQ ID NO: 245 is the determined cDNA sequence for JPTPN51

SEQ ID NO: 246 is the determined cDNA sequence for JPTPN56

SEQ ID NO: 247 is the determined cDNA sequence for PTPN64

SEQ ID NO: 248 is the determined cDNA sequence for JPTPN65

SEQ ID NO: 249 is the determined cDNA sequence for JPTPN67

SEQ ID NO: 250 is the determined cDNA sequence for JPTPN76

SEQ ID NO: 251 is the determined cDNA sequence for JPTPN84

SEQ ID NO: 252 is the determined cDNA sequence for JPTPN85

SEQ ID NO: 253 is the determined cDNA sequence for JPTPN86

SEQ ID NO: 254 is the determined cDNA sequence for JPTPN87

SEQ ID NO: 255 is the determined cDNA sequence for JPTPN88

SEQ ID NO: 256 is the determined cDNA sequence for JP1 F1

SEQ ID NO: 257 is the determined cDNA sequence for JP1F2

SEQ ID NO: 258 is the determined cDNA sequence for JP1C2

SEQ ID NO: 259 is the determined cDNA sequence for JP1B1

SEQ ID NO: 260 is the determined cDNA sequence for JP1B2

SEQ ID NO: 261 is the determined cDNA sequence for JP1D3

SEQ ID NO: 262 is the determined cDNA sequence for JP1A4

SEQ ID NO: 263 is the determined cDNA sequence for JP1F5

SEQ ID NO: 264 is the determined cDNA sequence for JP1E6

SEQ ID NO: 265 is the determined cDNA sequence for JP1D6

SEQ ID NO: 266 is the determined cDNA sequence for JP1B5

SEQ ID NO: 267 is the determined cDNA sequence for JP1A6

SEQ ID NO: 268 is the determined cDNA sequence for JP1E8

SEQ ID NO: 269 is the determined cDNA sequence for JP1D7

SEQ ID NO: 270 is the determined cDNA sequence for JP1D9

SEQ ID NO: 271 is the determined cDNA sequence for JP1C10

SEQ ID NO: 272 is the determined cDNA sequence for JP1A9

SEQ ID NO: 273 is the determined cDNA sequence for JP1F12

SEQ ID NO: 274 is the determined cDNA sequence for JP1E12

SEQ ID NO: 275 is the determined cDNA sequence for JP1D11

SEQ ID NO: 276 is the determined cDNA sequence for JP1C11

SEQ ID NO: 277 is the determined cDNA sequence for JP1C12

SEQ ID NO: 278 is the determined cDNA sequence for JP1B12

SEQ ID NO: 279 is the determined cDNA sequence for JP1A12

SEQ ID NO: 280 is the determined cDNA sequence for JP8G2

SEQ ID NO: 281 is the determined cDNA sequence for JP8H1

SEQ ID NO: 282 is the determined cDNA sequence for JP8H2

SEQ ID NO: 283 is the determined cDNA sequence for JP8A3

SEQ ID NO: 284 is the determined cDNA sequence for JP8A4

SEQ ID NO: 285 is the determined cDNA sequence for JP8C3

SEQ ID NO: 286 is the determined cDNA sequence for JP8G4

SEQ ID NO: 287 is the determined cDNA sequence for JP8B6

SEQ ID NO: 288 is the determined cDNA sequence for JP8D6

SEQ ID NO: 289 is the determined cDNA sequence for JP8F5

SEQ ID NO: 290 is the determined cDNA sequence for JP8A8

SEQ ID NO: 291 is the determined cDNA sequence for JP8C7

SEQ ID NO: 292 is the determined cDNA sequence for JP8D7

SEQ ID NO: 293 is the determined cDNA sequence for P8D8

SEQ ID NO: 294 is the determined cDNA sequence for JP8E7

SEQ ID NO: 295 is the determined cDNA sequence for JP8F8

SEQ ID NO: 296 is the determined cDNA sequence for JP8G8

SEQ ID NO: 297 is the determined cDNA sequence for JP8B10

SEQ ID NO: 298 is the determined cDNA sequence for JP8C10

SEQ ID NO: 299 is the determined cDNA sequence for JP8E9

SEQ ID NO: 300 is the determined cDNA sequence for JP8E10

SEQ ID NO: 301 is the determined cDNA sequence for JP8F9

SEQ ID NO: 302 is the determined cDNA sequence for JP8H9

SEQ ID NO: 303 is the determined cDNA sequence for JP8C12

SEQ ID NO: 304 is the determined cDNA sequence for JP8E11

SEQ ID NO: 305 is the determined cDNA sequence for JP8E12

SEQ ID NO: 306 is the amino acid sequence for the peptide PS2#12

SEQ ID NO: 307 is the determined cDNA sequence for P711P

SEQ ID NO: 308 is the determined cDNA sequence for P712P

SEQ ID NO: 309 is the determined cDNA sequence for CLONE23

SEQ ID NO: 310 is the determined cDNA sequence for P774P

SEQ ID NO: 311 is the determined cDNA sequence for P775P

SEQ ID NO: 312 is the determined cDNA sequence for P715P

SEQ ID NO: 313 is the determined cDNA sequence for P710P

SEQ ID NO: 314 is the determined cDNA sequence for P767P

SEQ ID NO: 315 is the determined cDNA sequence for P768P

SEQ ID NO: 316-325 are the determined cDNA sequences of previouslyisolated genes

SEQ ID NO: 326 is the determined cDNA sequence for P703PDE5

SEQ ID NO: 327 is the amino acid sequence for P703PDE5

SEQ ID NO: 328 is the determined cDNA sequence for P703P6.26

SEQ ID NO: 329 is the amino acid sequence for P703P6.26

SEQ ID NO: 330 is the determined cDNA sequence for P703PX-23

SEQ ID NO: 331 is the amino acid sequence for P703PX-23

SEQ ID NO: 332 is the determined full length cDNA sequence for P509S

SEQ ID NO: 333 is the determined extended cDNA sequence for P707P (alsoreferred to as 11-C9)

SEQ ID NO: 334 is the determined cDNA sequence for P714P

SEQ ID NO: 335 is the determined cDNA sequence for P705P (also referredto as 9-F3)

SEQ ID NO: 336 is the amino acid sequence for P705P

SEQ ID NO: 337 is the amino acid sequence of the peptide P1S#10

SEQ ID NO: 338 is the amino acid sequence of the peptide p5

SEQ ID NO: 339 is the amino acid sequence of P509S

SEQ ID NO: 340 is the determined cDNA sequence for P778P

SEQ ID NO: 341 is the determined cDNA sequence for P786P

SEQ ID NO: 342 is the determined cDNA sequence for P789P

SEQ ID NO: 343 is the determined cDNA sequence for a clone showinghomology to Homo sapiens MM46 mRNA

SEQ ID NO: 344 is the determined cDNA sequence for a clone showinghomology to Homo sapiens TNF-alpha stimulated ABC protein (ABC50) mRNA

SEQ ID NO: 345 is the determined cDNA sequence for a clone showinghomology to Homo sapiens mRNA for E-cadherin

SEQ ID NO: 346 is the determined cDNA sequence for a clone showinghomology to Human nuclear-encoded mitochondrial serinehydroxymethyltransferase (SHMT)

SEQ ID NO: 347 is the determined cDNA sequence for a clone showinghomology to Homo sapiens natural resistance-associated macrophageprotein2 (NRAMP2)

SEQ ID NO: 348 is the determined cDNA sequence for a clone showinghomology to Homo sapiens phosphoglucomutase-related protein (PGMRP)

SEQ ID NO: 349 is the determined cDNA sequence for a clone showinghomology to Human mRNA for proteosome subunit p40

SEQ ID NO: 350 is the determined cDNA sequence for P777P

SEQ ID NO: 351 is the determined cDNA sequence for P779P

SEQ ID NO: 352 is the determined cDNA sequence for P790P

SEQ ID NO: 353 is the determined cDNA sequence for P784P

SEQ ID NO: 354 is the determined cDNA sequence for P776P

SEQ ID NO: 355 is the determined cDNA sequence for P780P

SEQ ID NO: 356 is the determined cDNA sequence for P544S

SEQ ID NO: 357 is the determined cDNA sequence for P745S

SEQ ID NO: 358 is the determined cDNA sequence for P782P

SEQ ID NO: 359 is the determined cDNA sequence for P783P

SEQ ID NO: 360 is the determined cDNA sequence for unknown 17984

SEQ ID NO: 361 is the determined cDNA sequence for P787P

SEQ ID NO: 362 is the determined cDNA sequence for P788P

SEQ ID NO: 363 is the determined cDNA sequence for unknown 17994

SEQ ID NO: 364 is the determined cDNA sequence for P781P

SEQ ID NO: 365 is the determined cDNA sequence for P785P

SEQ ID NO: 366-375 are the determined cDNA sequences for splice variantsof B305D.

SEQ ID NO: 376 is the amino acid sequence encoded by the sequence of SEQID NO: 366.

SEQ ID NO: 377 is the amino acid sequence encoded by the sequence of SEQID NO: 372.

SEQ ID NO: 378 is the amino acid sequence encoded by the sequence of SEQID NO: 373.

SEQ ID NO: 379 is the amino acid sequence encoded by the sequence of SEQID NO: 374.

SEQ ID NO: 380 is the amino acid sequence encoded by the sequence of SEQID NO: 375.

SEQ ID NO: 381 is the determined cDNA sequence for B716P.

SEQ ID NO: 382 is the determined full-length cDNA sequence for P711P.

SEQ ID NO: 383 is the amino acid sequence for P711P.

SEQ ID NO: 384 is the cDNA sequence for P1000C.

SEQ ID NO: 385 is the cDNA sequence for CGI-82.

SEQ ID NO:386 is the cDNA sequence for 23320.

SEQ ID NO:387 is the cDNA sequence for CGI-69.

SEQ ID NO:388 is the cDNA sequence for L-iditol-2-dehydrogenase.

SEQ ID NO:389 is the cDNA sequence for 23379.

SEQ ID NO:390 is the cDNA sequence for 23381.

SEQ ID NO:391 is the cDNA sequence for KIAA0122.

SEQ ID NO:392 is the cDNA sequence for clone 23399 (also known asP554S).

SEQ ID NO:393 is the cDNA sequence for a previously identified gene.

SEQ ID NO:394 is the cDNA sequence for HCLBP.

SEQ ID NO:395 is the cDNA sequence for transglutaminase.

SEQ ID NO:396 is the cDNA sequence for a previously identified gene.

SEQ ID NO:397 is the cDNA sequence for PAP.

SEQ ID NO:398 is the cDNA sequence for Ets transcription factor PDEF.

SEQ ID NO:399 is the cDNA sequence for hTGR.

SEQ ID NO:400 is the cDNA sequence for KIAA0295.

SEQ ID NO:401 is the cDNA sequence for 22545.

SEQ ID NO:402 is the cDNA sequence for 22547.

SEQ ID NO:403 is the cDNA sequence for 22548.

SEQ ID NO:404 is the cDNA sequence for 22550.

SEQ ID NO:405 is the cDNA sequence for 22551.

SEQ ID NO:406 is the cDNA sequence for 22552.

SEQ ID NO:407 is the cDNA sequence for 22553 (also known as P1020C).

SEQ ID NO:408 is the cDNA sequence for 22558.

SEQ ID NO:409 is the cDNA sequence for 22562.

SEQ ID NO:410 is the cDNA sequence for 22565.

SEQ ID NO:411 is the cDNA sequence for 22567.

SEQ ID NO:412 is the cDNA sequence for 22568.

SEQ ID NO:413 is the cDNA sequence for 22570.

SEQ ID NO:414 is the cDNA sequence for 22571.

SEQ ID NO:415 is the cDNA sequence for 22572.

SEQ ID NO:416 is the cDNA sequence for 22573.

SEQ ID NO:417 is the cDNA sequence for 22573.

SEQ ID NO:418 is the cDNA sequence for 22575.

SEQ ID NO:419 is the cDNA sequence for 22580.

SEQ ID NO:420 is the cDNA sequence for 22581.

SEQ ID NO:421 is the cDNA sequence for 22582.

SEQ ID NO:422 is the cDNA sequence for 22583.

SEQ ID NO:423 is the cDNA sequence for 22584.

SEQ ID NO:424 is the cDNA sequence for 22585.

SEQ ID NO:425 is the cDNA sequence for 22586.

SEQ ID NO:426 is the cDNA sequence for 22587.

SEQ ID NO:427 is the cDNA sequence for 22588.

SEQ ID NO:428 is the cDNA sequence for 22589.

SEQ ID NO:429 is the cDNA sequence for 22590.

SEQ ID NO:430 is the cDNA sequence for 22591.

SEQ ID NO:431 is the cDNA sequence for 22592.

SEQ ID NO:432 is the cDNA sequence for 22593.

SEQ ID NO:433 is the cDNA sequence for 22594.

SEQ ID NO:434 is the cDNA sequence for 22595.

SEQ ID NO:435 is the cDNA sequence for 22596.

SEQ ID NO:436 is the cDNA sequence for 22847.

SEQ ID NO:437 is the cDNA sequence for 22848.

SEQ ID NO:438 is the cDNA sequence for 22849.

SEQ ID NO:439 is the cDNA sequence for 22851.

SEQ ID NO:440 is the cDNA sequence for 22852.

SEQ ID NO:441 is the cDNA sequence for 22853.

SEQ ID NO:442 is the cDNA sequence for 22854.

SEQ ID NO:443 is the cDNA sequence for 22855.

SEQ ID NO:444 is the cDNA sequence for 22856.

SEQ ID NO:445 is the cDNA sequence for 22857.

SEQ ID NO:446 is the cDNA sequence for 23601.

SEQ ID NO:447 is the cDNA sequence for 23602.

SEQ ID NO:448 is the cDNA sequence for 23605.

SEQ ID NO:449 is the cDNA sequence for 23606.

SEQ ID NO:450 is the cDNA sequence for 23612.

SEQ ID NO:451 is the cDNA sequence for 23614.

SEQ ID NO:452 is the cDNA sequence for 23618.

SEQ ID NO:453 is the cDNA sequence for 23622.

SEQ ID NO:454 is the cDNA sequence for folate hydrolase.

SEQ ID NO:455 is the cDNA sequence for LIM protein.

SEQ ID NO:456 is the cDNA sequence for a known gene.

SEQ ID NO:457 is the cDNA sequence for a known gene.

SEQ ID NO:458 is the cDNA sequence for a previously identified gene.

SEQ ID NO:459 is the cDNA sequence for 23045.

SEQ ID NO:460 is the cDNA sequence for 23032.

SEQ ID NO:461 is the cDNA sequence for clone 23054.

SEQ ID NO:462-467 are cDNA sequences for known genes.

SEQ ID NO:468-471 are cDNA sequences for P710P.

SEQ ID NO:472 is a cDNA sequence for P1001C.

SEQ ID NO: 473 is the determined cDNA sequence for a first splicevariant of P775P (referred to as 27505).

SEQ ID NO: 474 is the determined cDNA sequence for a second splicevariant of P775P (referred to as 19947).

SEQ ID NO: 475 is the determined cDNA sequence for a third splicevariant of P775P (referred to as 19941).

SEQ ID NO: 476 is the determined cDNA sequence for a fourth splicevariant of P775P (referred to as 19937).

SEQ ID NO: 477 is a first amino acid sequence encoded by the sequence ofSEQ ID NO: 474.

SEQ ID NO: 478 is a second amino acid sequence encoded by the sequenceof SEQ ID NO: 474.

SEQ ID NO: 479 is the amino acid sequence encoded by the sequence of SEQID NO: 475.

SEQ ID NO: 480 is a first amino acid sequence encoded by the sequence ofSEQ ID NO: 473.

SEQ ID NO: 481 is a second amino acid sequence encoded by the sequenceof SEQ ID NO: 473.

SEQ ID NO: 482 is a third amino acid sequence encoded by the sequence ofSEQ ID NO: 473.

SEQ ID NO: 483 is a fourth amino acid sequence encoded by the sequenceof SEQ ID NO: 473.

SEQ ID NO: 484 is the first 30 amino acids of the M. tuberculosisantigen Ra12.

SEQ ID NO: 485 is the PCR primer AW025.

SEQ ID NO: 486 is the PCR primer AW003.

SEQ ID NO: 487 is the PCR primer AW027.

SEQ ID NO: 488 is the PCR primer AW026.

SEQ ID NO: 489-501 are peptides employed in epitope mapping studies.

SEQ ID NO: 502 is the determined cDNA sequence of the complementaritydetermining region for the anti-P503S monoclonal antibody 20D4.

SEQ ID NO: 503 is the determined cDNA sequence of the complementaritydetermining region for the anti-P503S monoclonal antibody JA1.

SEQ ID NO: 504 & 505 are peptides employed in epitope mapping studies.

SEQ ID NO: 506 is the determined cDNA sequence of the complementaritydetermining region for the anti-P703P monoclonal antibody 8H2.

SEQ ID NO: 507 is the determined cDNA sequence of the complementaritydetermining region for the anti-P703P monoclonal antibody 7H8.

SEQ ID NO: 508 is the determined cDNA sequence of the complementaritydetermining region for the anti-P703P monoclonal antibody 2D4.

SEQ ID NO: 509-522 are peptides employed in epitope mapping studies.

SEQ ID NO: 523 is a mature form of P703P used to raise antibodiesagainst P703P.

SEQ ID NO: 524 is the putative full-length cDNA sequence of P703P.

SEQ ID NO: 525 is the amino acid sequence encoded by SEQ ID NO: 524.

SEQ ID NO: 526 is the full-length cDNA sequence for P790P.

SEQ ID NO: 527 is the amino acid sequence for P790P.

SEQ ID NO: 528 & 529 are PCR primers.

SEQ ID NO: 530 is the cDNA sequence of a splice variant of SEQ ID NO:366.

SEQ ID NO: 531 is the cDNA sequence of the open reading frame of SEQ IDNO: 530.

SEQ ID NO: 532 is the amino acid encoded by the sequence of SEQ ID NO:531.

SEQ ID NO: 533 is the DNA sequence of a putative ORF of P775P.

SEQ ID NO: 534 is the amino acid sequence encoded by SEQ ID NO: 533.

SEQ ID NO: 535 is a first full-length cDNA sequence for P510S.

SEQ ID NO: 536 is a second full-length cDNA sequence for P510S.

SEQ ID NO: 537 is the amino acid sequence encoded by SEQ ID NO: 535.

SEQ ID NO: 538 is the amino acid sequence encoded by SEQ ID NO: 536.

SEQ ID NO: 539 is the peptide P501S-370.

SEQ ID NO: 540 is the peptide P501S-376.

SEQ ID NO: 541-551 are epitopes of P501S.

SEQ ID NO: 552 is an extended cDNA sequence for P712P.

SEQ ID NO: 553-568 are the amino acid sequences encoded by open readingframes within SEQ ID NO: 552.

SEQ ID NO: 569 is an extended cDNA sequence for P776P.

SEQ ID NO: 570 is the determined cDNA sequence for a splice variant ofP776P referred to as contig 6.

SEQ ID NO: 571 is the determined cDNA sequence for a splice variant ofP776P referred to as contig 7.

SEQ ID NO: 572 is the determined cDNA sequence for a splice variant ofP776P referred to as contig 14.

SEQ ID NO: 573 is the amino acid sequence encoded by a first ORF of SEQID NO: 570.

SEQ ID NO: 574 is the amino acid sequence encoded by a second ORF of SEQID NO: 570.

SEQ ID NO: 575 is the amino acid sequence encoded by a ORF of SEQ ID NO:571.

SEQ ID NO: 576-586 are amino acid sequences encoded by ORFs of SEQ IDNO: 569.

SEQ ID NO: 587 is a DNA consensus sequence of the sequences of P767P andP777P.

SEQ ID NO: 588-590 are amino acid sequences encoded by ORFs of SEQ IDNO: 587.

SEQ ID NO: 591 is an extended cDNA sequence for P1020C.

SEQ ID NO: 592 is the amino acid sequence encoded by the sequence of SEQID NO: P1020C.

SEQ ID NO: 593 is a splice variant of P775P referred to as 50748.

SEQ ID NO: 594 is a splice variant of P775P referred to as 50717. SEQ IDNO: 595 is a splice variant of P775P referred to as 45985.

SEQ ID NO: 596 is a splice variant of P775P referred to as 38769.

SEQ ID NO: 597 is a splice variant of P775P referred to as 37922.

SEQ ID NO: 598 is a splice variant of P510S referred to as 49274.

SEQ ID NO: 599 is a splice variant of P510S referred to as 39487.

SEQ ID NO: 600 is a splice variant of P504S referred to as 5167.16.

SEQ ID NO: 601 is a splice variant of P504S referred to as 5167.1.

SEQ ID NO: 602 is a splice variant of P504S referred to as 5163.46.

SEQ ID NO: 603 is a splice variant of P504S referred to as 5163.42.

SEQ ID NO: 604 is a splice variant of P504S referred to as 5163.34.

SEQ ID NO: 605 is a splice variant of P504S referred to as 5163.17.

SEQ ID NO: 606 is a splice variant of P501S referred to as 10640.

SEQ ID NO: 607-615 are the sequences of PCR primers.

SEQ ID NO: 616 is the determined cDNA sequence of a fusion of P703P andPSA.

SEQ ID NO: 617 is the amino acid sequence of the fusion of P703P andPSA.

SEQ ID NO: 618-689 are determined cDNA sequences of prostate-specificclones.

SEQ ID NO: 690 is the cDNA sequence of the gene DD3.

SEQ ID NO: 691-697 are determined cDNA sequences of prostate-specificclones.

SEQ ID NO: 698 is an extended cDNA sequence for P714P.

SEQ ID NO: 699-701 are the cDNA sequences for splice variants of P704P.

SEQ ID NO: 702 is the cDNA sequence of a spliced variant of P553Sreferred to as P553S-14.

SEQ ID NO: 703 is the cDNA sequence of a spliced variant of P553Sreferred to as P553S-12.

SEQ ID NO: 704 is the cDNA sequence of a spliced variant of P553Sreferred to as P553S-10.

SEQ ID NO: 705 is the cDNA sequence of a spliced variant of P553Sreferred to as P553S-6.

SEQ ID NO: 706 is the amino acid sequence encoded by SEQ ID NO: 705.

SEQ ID NO: 707 is the amino acid sequence encoded by SEQ ID NO: 702 SEQID NO: 708 is a second amino acid sequence encoded by SEQ ID NO: 702.

SEQ ID NO: 709-772 are determined cDNA sequences of prostate-specificclones.

SEQ ID NO: 773 is a first full-length cDNA sequence forprostate-specific transglutaminase gene (also referred to herein asP558S).

SEQ ID NO: 774 is a second full-length cDNA sequence forprostate-specific transglutaminase gene.

SEQ ID NO: 775 is the amino acid sequence encoded by the sequence of SEQID NO: 773.

SEQ ID NO: 776 is the amino acid sequence encoded by the sequence of SEQID NO: 774.

SEQ ID NO: 777 is the full-length cDNA sequence for P788P.

SEQ ID NO: 778 is the amino acid sequence encoded by SEQ ID NO: 777.

SEQ ID NO: 779 is the determined cDNA sequence for a polymorphic variantof P788P.

SEQ ID NO: 780 is the amino acid sequence encoded by SEQ ID NO: 779.

SEQ ID NO: 781 is the amino acid sequence of peptide 4 from P703P.

SEQ ID NO: 782 is the cDNA sequence that encodes peptide 4 from P703P.

SEQ ID NO: 783-798 are the cDNA sequence encoding epitopes of P703P.

SEQ ID NO: 799-814 are the amino acid sequences of epitopes of P703P.

SEQ ID NO: 815 and 816 are PCR primers.

SEQ ID NO: 817 is the cDNA sequence encoding an N-terminal portion ofP788P expressed in E. coli.

SEQ ID NO: 818 is the amino acid sequence of the N-terminal portion ofP788P expressed in E. coli.

SEQ ID NO: 819 is the amino acid sequence of the M. tuberculosis antigenRa12.

SEQ ID NO: 820 and 821 are PCR primers.

SEQ ID NO: 822 is the cDNA sequence for the Ra12-P510S-C construct.

SEQ ID NO: 823 is the cDNA sequence for the P510S-C construct.

SEQ ID NO: 824 is the cDNA sequence for the P510S-E3 construct.

SEQ ID NO: 825 is the amino acid sequence for the Ra12-P510S-Cconstruct.

SEQ ID NO: 826 is the amino acid sequence for the P510S-C construct.

SEQ ID NO: 827 is the amino acid sequence for the P510S-E3 construct.

SEQ ID NO: 828-833 are PCR primers.

SEQ ID NO: 834 is the cDNA sequence of the construct Ra12-P775P-ORF3.

SEQ ID NO: 835 is the amino acid sequence of the constructRa12-P775P-ORF3.

SEQ ID NO: 836 and 837 are PCR primers.

SEQ ID NO: 838 is the determined amino acid sequence for a P703P His tagfusion protein.

SEQ ID NO: 839 is the determined cDNA sequence for a P703P His tagfusion protein.

SEQ ID NO: 840 and 841 are PCR primers.

SEQ ID NO: 842 is the determined amino acid sequence for a P705P His tagfusion protein.

SEQ ID NO: 843 is the determined cDNA sequence for a P705P His tagfusion protein.

SEQ ID NO: 844 and 845 are PCR primers.

SEQ ID NO: 846 is the determined amino acid sequence for a P711P His tagfusion protein.

SEQ ID NO: 847 is the determined cDNA sequence for a P711P His tagfusion protein.

SEQ ID NO: 848 is the amino acid sequence of the M. tuberculosis antigenRa12.

SEQ ID NO: 849 and 850 are PCR primers.

SEQ ID NO: 851 is the determined cDNA sequence for the constructRa12-P501S-E2.

SEQ ID NO: 852 is the determined amino acid sequence for the constructRa12-P501S-E2.

SEQ ID NO: 853 is the amino acid sequence for an epitope of P501S.

SEQ ID NO: 854 is the DNA sequence encoding SEQ ID NO: 853.

SEQ ID NO: 855 is the amino acid sequence for an epitope of P501S.

SEQ ID NO: 856 is the DNA sequence encoding SEQ ID NO: 855.

SEQ ID NO: 857 is a peptide employed in epitope mapping studies.

SEQ ID NO: 858 is the amino acid sequence for an epitope of P501S.

SEQ ID NO: 859 is the DNA sequence encoding SEQ ID NO: 858.

SEQ ID NO: 860-862 are the amino acid sequences for CD4 epitopes ofP501S.

SEQ ID NO: 863-865 are the DNA sequences encoding the sequences of SEQID NO: 860-862.

SEQ ID NO: 866-877 are the amino acid sequences for putative CTLepitopes of P703P.

SEQ ID NO: 878 is the full-length cDNA sequence for P789P.

SEQ ID NO: 879 is the amino acid sequence encoded by SEQ ID NO: 878.

SEQ ID NO: 880 is the determined full-length cDNA sequence for thesplice variant of P776P referred to as contig 6.

SEQ ID NO: 881-882 are determined full-length cDNA sequences for thesplice variant of P776P referred to as contig 7.

SEQ ID NO: 883-887 are amino acid sequences encoded by SEQ ID NO: 880.

SEQ ID NO: 888-893 are amino acid sequences encoded by the splicevariant of P776P referred to as contig 7.

SEQ ID NO: 894 is the full-length cDNA sequence for human transmembraneprotease serine 2.

SEQ ID NO: 895 is the amino acid sequence encoded by SEQ ID NO: 894.

SEQ ID NO: 896 is the cDNA sequence encoding the first 209 amino acidsof human transmembrane protease serine 2.

SEQ ID NO: 897 is the first 209 amino acids of human transmembraneprotease serine 2.

SEQ ID NO: 898 is the amino acid sequence of peptide 296-322 of P501S.

SEQ ID NO: 899-902 are PCR primers.

SEQ ID NO: 903 is the determined cDNA sequence of the Vb chain of a Tcell receptor for the P501S-specific T cell clone 4E5.

SEQ ID NO: 904 is the determined cDNA sequence of the Va chain of a Tcell receptor for the P501S-specific T cell clone 4E5.

SEQ ID NO: 905 is the amino acid sequence encoded by SEQ ID NO 903.

SEQ ID NO: 906 is the amino acid sequence encoded by SEQ ID NO 904.

SEQ ID NO: 907 is the full-length open reading frame for P768P includingstop codon.

SEQ ID NO: 908 is the full-length open reading frame for P768P withoutstop codon.

SEQ ID NO: 909 is the amino acid sequence encoded by SEQ ID NO: 908.

SEQ ID NO: 910-915 are the amino acid sequences for predicted domains ofP768P.

SEQ ID NO: 916 is the full-length cDNA sequence of P835P.

SEQ ID NO: 917 is the cDNA sequence of the previously identified cloneFLJ13581.

SEQ ID NO: 918 is the cDNA sequence of the open reading frame for P835Pwith stop codon.

SEQ ID NO: 919 is the cDNA sequence of the open reading frame for P835Pwithout stop codon.

SEQ ID NO: 920 is the full-length amino acid sequence for P835P.

SEQ ID NO: 921-928 are the amino acid sequences of extracellular andintracellular domains of P835P.

SEQ ID NO: 929 is the full-length cDNA sequence for P1000C.

SEQ ID NO: 930 is the cDNA sequence of the open reading frame forP1000C, including stop codon.

SEQ ID NO: 931 is the cDNA sequence of the open reading frame forP1000C, without stop codon.

SEQ ID NO: 932 is the full-length amino acid sequence for P1000C.

SEQ ID NO: 933 is amino acids 1-100 of SEQ ID NO: 932.

SEQ ID NO: 934 is amino acids 100-492 of SEQ ID NO: 932.

SEQ ID NO: 935-937 are PCR primers.

SEQ ID NO: 938 is the cDNA sequence of the expressed full-length P767Pcoding region.

SEQ ID NO: 939 is the cDNA sequence of an expressed truncated P767Pcoding region.

SEQ ID NO: 940 is the amino acid sequence encoded by SEQ ID NO: 939.

SEQ ID NO: 941 is the amino acid sequence encoded by SEQ ID NO: 938.

SEQ ID NO: 942 is the DNA sequence of a CD4 epitope of P703P.

SEQ ID NO: 943 is the amino acid sequence of a CD4 epitope of P703P.

SEQ ID NO: 944 and 945 are the cDNA sequences of two alternative spliceforms of P780P.

SEQ ID NO: 946 and 947 are the amino acid sequences encoded by SEQ IDNO: 944 and 945, respectively.

SEQ ID NO: 948 is a corrected cDNA sequence for P705P.

SEQ ID NO: 949-956 are the amino acid sequences of P790P peptides.

SEQ ID NO: 957-966 are the amino acid sequences of P775P peptides.

SEQ ID NO: 967 is an extended cDNA sequence for P554S.

SEQ ID NO: 968 is the amino acid sequence for P554S.

SEQ ID NO: 969-976 are the cDNA sequences encoding epitopes of P501S.

SEQ ID NO: 977-984 are the amino acid sequences of epitopes of P501S.

SEQ ID NO: 985-987 are amino acid sequences of peptides of P501S.

SEQ ID NO: 988-990 are amino acid sequences of peptides of P703P.

SEQ ID NO:991 is the cDNA encoding a CD4+ T cell epitope of P703P.

SEQ ID NO:992 is the amino acid sequence of a CD4+ T cell epitope ofP703P (encoded by SEQ ID NO:991).

SEQ ID NO: 993 represents the cDNA sequence for P706P clone 8-B6 thatincludes P3.

SEQ ID NO: 994 represents the cDNA sequence for P706P clone 8-B6 thatincludes P4.

SEQ ID NO: 995 represents the cDNA sequence for P706Pclone 8B6.P4contig.

SEQ ID NO: 996 represents the cDNA sequence for P706P ESTs 1386-1902.

SEQ ID NO: 997 represents the cDNA sequence for P706P ESTs 4927-6088.

SEQ ID NO: 998 represents the cDNA sequence for P713P clone PT4410A42.

SEQ ID NO: 999-1002 represent the four exons encoding the prostaticsecretory protein (PSP).

SEQ ID NO: 1003 represents the full length amino acid sequence for PSP.

SEQ ID NO: 1004 represents an extended cDNA sequence for clone P1E.

SEQ ID NO: 1005 represents the amino acid sequence of an open readingframe encoded by SEQ ID NO: 1004.

SEQ ID NO: 1006 represents the DNA sequence of the coding region of theexpression construct P510.seq.

SEQ ID NO: 1007 represents the DNA sequence of coding region of theexpression construct MAPS-P510S.seq.

SEQ ID NO: 1008 represents the protein sequence encoded by the DNAsequence of SEQ ID NO: 1007.

SEQ ID NO: 1009 represents the protein sequence encoded by the DNAsequence of SEQ ID NO: 1006.

SEQ ID NO: 1010 represents the DNA sequence of the coding region for aTrx-P501S fusion construct.

SEQ ID NO: 1011 represents the amino acid sequence for a Trx-P501Sfusion protein that is encoded by the DNA sequence of SEQ ID NO: 1010.

SEQ ID NO:1012 represents the amino acid sequence for a minimal P501Sepitope recognized by clone 2H2-1A12.

SEQ ID NO:1013 represents the DNA sequence for a minimal P501S epitoperecognized by clone 2H2-1A12.

SEQ ID NO:1014 corresponds to the primer sequence for the forward primerhPAPF1.

SEQ ID NO:1015 corresponds to the primer sequence for the reverse primerhPAPRV1.

SEQ ID NO:1016 corresponds to the primer sequence for the forward primerFOPP2F1.

SEQ ID NO:1017 corresponds to the primer sequence for the reverse primerFOPPF1.

SEQ ID NO:1018 corresponds to the primer sequence for the forward primerFOPP2RV1.

SEQ ID NO:1019 sets forth a DNA sequence for a fusion of FOPP and hPAP,referred to as FOPP2.

SEQ ID NO:1020 sets forth an amino acid sequence for a fusion of FOPPand hPAP, referred to as FOPP2.

SEQ ID NO:1021 sets forth a DNA sequence for a P501S minimal CD8+ T cellepitope recognized by clone 1H1-1A6.

SEQ ID NO:1022 sets forth an amino acid sequence for a P501S minimalCD8+ T cell epitope recognized by clone 1H1-1A6.

SEQ ID NO:1023 sets forth the sequence for the primer PDM-930.

SEQ ID NO:1024 sets forth the sequence for the primer PDM-165.

SEQ ID NO:1025 sets forth the sequence for the primer PDM-929.

SEQ ID NO1026: sets forth a DNA sequence encoding the P501S D protein(amino acids 316-553).

SEQ ID NO:1027 sets forth a DNA sequence encoding the P501S C protein(amino acids 257-553).

SEQ ID NO:1028 sets forth an amino acid sequence corresponding to theP501S D protein (amino acids 316-553).

SEQ ID NO:1029 sets forth an amino acid sequence corresponding to theP501 S C protein (amino acids 257-553).

SEQ ID NO:1030 sets forth an amino acid for a P703P epitope recognizedby patient sera.

SEQ ID NO:1031 sets forth an amino acid for a P703P epitope recognizedby patient sera.

SEQ ID NO:1032 sets forth an amino acid for a P703P epitope recognizedby patient sera.

SEQ ID NO:1033 sets forth an amino acid for a P703P epitope recognizedby patient sera.

SEQ ID NO:1034 discloses an additional DNA sequence for the prostateantigen P712P.

SEQ ID NO:1035 discloses an additional DNA sequence for the prostateantigen P775P.

SEQ ID NO:1036 discloses an additional DNA sequence for the prostateantigen P704P.

SEQ ID NO:1037 discloses an 11 amino acid fragment derived from P501Sthat contains naturally processed epitopes for at least three class Ialleles.

SEQ ID NO:1038 discloses the polynucleotide sequence encoding SEQ ID NO:1037.

DETAILED DESCRIPTION OF THE INVENTION

U.S. patents, U.S. patent application publications, U.S. patentapplications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification and/or listedin the Application Data Sheet, are incorporated herein by reference, intheir entirety.

The present invention is directed generally to compositions and theiruse in the therapy and diagnosis of cancer, particularly prostatecancer. As described further below, illustrative compositions of thepresent invention include, but are not restricted to, polypeptides,particularly immunogenic polypeptides, polynucleotides encoding suchpolypeptides, antibodies and other binding agents, antigen presentingcells (APCs) and immune system cells (e.g., T cells).

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of virology,immunology, microbiology, molecular biology and recombinant DNAtechniques within the skill of the art, many of which are describedbelow for the purpose of illustration. Such techniques are explainedfully in the literature. See, e.g., Sambrook, et al. Molecular Cloning:A Laboratory Manual (2nd Edition, 1989); Maniatis et al. MolecularCloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach,vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed.,1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guideto Molecular Cloning (1984).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

Polypeptide Compositions

As used herein, the term “polypeptide” is used in its conventionalmeaning, i.e., as a sequence of amino acids. The polypeptides are notlimited to a specific length of the product; thus, peptides,oligopeptides, and proteins are included within the definition ofpolypeptide, and such terms may be used interchangeably herein unlessspecifically indicated otherwise. This term also does not refer to orexclude post-expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like, as well asother modifications known in the art, both naturally occurring andnon-naturally occurring. A polypeptide may be an entire protein, or asubsequence thereof. Particular polypeptides of interest in the contextof this invention are amino acid subsequences comprising epitopes, i.e.,antigenic determinants substantially responsible for the immunogenicproperties of a polypeptide and being capable of evoking an immuneresponse.

Particularly illustrative polypeptides of the present invention comprisethose encoded by a polynucleotide sequence set forth in any one of SEQID NO: 1-111, 115-171, 173-175, 177,179-305, 307-315, 326, 328, 330,332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535,536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817,823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939,942, 944, 945, 948, 967, 969-976, 991, 993-1002, 1004, 1006, 1007, 1010,1013-1019, 1021, 1023-1027 and 1034-1036 or a sequence that hybridizesunder moderately stringent conditions, or, alternatively, under highlystringent conditions, to a polynucleotide sequence set forth in any oneof SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305, 307-315, 326, 328,330, 332-335, 340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533,535, 536, 552, 569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789,817, 823, 824, 878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938,939, 942, 944, 945, 948, 967, 969-976, 991, 993-1002, 1004, 1006, 1007,1010, 1013-1019, 1021, 1023-1027 and 1034-1036. In specific embodiments,the polypeptides of the invention comprise amino acid sequences as setforth in any one of SEQ ID NO: 112-114, 172, 176, 178, 327, 329, 331,336, 339, 376-380, 383, 477-483, 496, 504, 505, 519, 520, 522, 525, 527,532, 534, 537-551, 553-568, 573-586, 588-590, 592, 706-708, 775, 776,778, 780, 781, 811, 814, 818, 826, 827, 853, 855, 858, 860-862, 866-877,879, 883-893, 895, 897, 898, 909-915, 920-928, 932-934, 940, 941, 943,946, 947, 949-966, 968, 977-990, 992, 1003, 1005, 1008, 1009, 1011, and1012, 1020, 1022, 1028-1029, 1030-1033, and 1037.

The polypeptides of the present invention are sometimes herein referredto as prostate-specific proteins or prostate-specific polypeptides, asan indication that their identification has been based at least in partupon their increased levels of expression in prostate tissue samples.Thus, a “prostate-specific polypeptide” or “prostate-specific protein,”refers generally to a polypeptide sequence of the present invention, ora polynucleotide sequence encoding such a polypeptide, that is expressedin a substantial proportion of prostate tissue samples, for examplepreferably greater than about 20%, more preferably greater than about30%, and most preferably greater than about 50% or more of prostatetissue samples tested, at a level that is at least two fold, andpreferably at least five fold, greater than the level of expression inother normal tissues, as determined using a representative assayprovided herein. A prostate-specific polypeptide sequence of theinvention, based upon its increased level of expression in tumor cells,has particular utility both as a diagnostic marker as well as atherapeutic target, as further described below.

In certain preferred embodiments, the polypeptides of the invention areimmunogenic, i.e., they react detectably within an immunoassay (such asan ELISA or T-cell stimulation assay) with antisera and/or T-cells froma patient with prostate cancer. Screening for immunogenic activity canbe performed using techniques well known to the skilled artisan. Forexample, such screens can be performed using methods such as thosedescribed in Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988. In one illustrative example, apolypeptide may be immobilized on a solid support and contacted withpatient sera to allow binding of antibodies within the sera to theimmobilized polypeptide. Unbound sera may then be removed and boundantibodies detected using, for example, ¹²⁵I-labeled Protein A.

As would be recognized by the skilled artisan, immunogenic portions ofthe polypeptides disclosed herein are also encompassed by the presentinvention. An “immunogenic portion,” as used herein, is a fragment of animmunogenic polypeptide of the invention that itself is immunologicallyreactive (i.e., specifically binds) with the B-cells and/or T-cellsurface antigen receptors that recognize the polypeptide. Immunogenicportions may generally be identified using well known techniques, suchas those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247(Raven Press, 1993) and references cited therein. Such techniquesinclude screening polypeptides for the ability to react withantigen-specific antibodies, antisera and/or T-cell lines or clones. Asused herein, antisera and antibodies are “antigen-specific” if theyspecifically bind to an antigen (i.e., they react with the protein in anELISA or other immunoassay, and do not react detectably with unrelatedproteins). Such antisera and antibodies may be prepared as describedherein, and using well-known techniques.

In one preferred embodiment, an immunogenic portion of a polypeptide ofthe present invention is a portion that reacts with antisera and/orT-cells at a level that is not substantially less than the reactivity ofthe full-length polypeptide (e.g., in an ELISA and/or T-cell reactivityassay). Preferably, the level of immunogenic activity of the immunogenicportion is at least about 50%, preferably at least about 70% and mostpreferably greater than about 90% of the immunogenicity for thefull-length polypeptide. In some instances, preferred immunogenicportions will be identified that have a level of immunogenic activitygreater than that of the corresponding full-length polypeptide, e.g.,having greater than about 100% or 150% or more immunogenic activity.

In certain other embodiments, illustrative immunogenic portions mayinclude peptides in which an N-terminal leader sequence and/ortransmembrane domain has been deleted. Other illustrative immunogenicportions will contain a small N- and/or C-terminal deletion (e.g., 1-30amino acids, preferably 5-15 amino acids), relative to the matureprotein.

In another embodiment, a polypeptide composition of the invention mayalso comprise one or more polypeptides that are immunologically reactivewith T cells and/or antibodies generated against a polypeptide of theinvention, particularly a polypeptide having an amino acid sequencedisclosed herein, or to an immunogenic fragment or variant thereof.

In another embodiment of the invention, polypeptides are provided thatcomprise one or more polypeptides that are capable of eliciting T cellsand/or antibodies that are immunologically reactive with one or morepolypeptides described herein, or one or more polypeptides encoded bycontiguous nucleic acid sequences contained in the polynucleotidesequences disclosed herein, or immunogenic fragments or variantsthereof, or to one or more nucleic acid sequences which hybridize to oneor more of these sequences under conditions of moderate to highstringency.

The present invention, in another aspect, provides polypeptide fragmentscomprising at least about 5, 10, 15, 20, 25, 50, or 100 contiguous aminoacids, or more, including all intermediate lengths, of a polypeptidecomposition set forth herein, such as those set forth in SEQ ID NO:112-114, 172, 176, 178, 327, 329, 331, 336, 339, 376-380, 383, 477-483,496, 504, 505, 519, 520, 522, 525, 527, 532, 534, 537-551, 553-568,573-586, 588-590, 592, 706-708, 775, 776, 778, 780, 781, 811, 814, 818,826, 827, 853, 855, 858, 860-862, 866-877, 879, 883-893, 895, 897, 898,909-915, 920-928, 932-934, 940, 941, 943, 946, 947, 949-966, 968,977-990, 992, 1003, 1005, 1008, 1009, 1011, 1012, 1020, 1022, 1028-1029,1030-1033, and 1033 or those encoded by a polynucleotide sequence setforth in a sequence of SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305,307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524,526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705,709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908,916-919, 929-931, 938, 939, 942, 944, 945, 948, 967, 969-976, 991,993-1002, 1004, 1006, 1007, 1010, 1013-1019, 1021, 1023-1027 and1034-1036.

In another aspect, the present invention provides variants of thepolypeptide compositions described herein. Polypeptide variantsgenerally encompassed by the present invention will typically exhibit atleast about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more identity (determined as described below), along itslength, to a polypeptide sequence set forth herein.

In one preferred embodiment, the polypeptide fragments and variantsprovided by the present invention are immunologically reactive with anantibody and/or T-cell that reacts with a full-length polypeptidespecifically set forth herein.

In another preferred embodiment, the polypeptide fragments and variantsprovided by the present invention exhibit a level of immunogenicactivity of at least about 50%, preferably at least about 70%, and mostpreferably at least about 90% or more of that exhibited by a full-lengthpolypeptide sequence specifically set forth herein.

A polypeptide “variant,” as the term is used herein, is a polypeptidethat typically differs from a polypeptide specifically disclosed hereinin one or more substitutions, deletions, additions and/or insertions.Such variants may be naturally occurring or may be syntheticallygenerated, for example, by modifying one or more of the abovepolypeptide sequences of the invention and evaluating their immunogenicactivity as described herein using any of a number of techniques wellknown in the art.

For example, certain illustrative variants of the polypeptides of theinvention include those in which one or more portions, such as anN-terminal leader sequence or transmembrane domain, have been removed.Other illustrative variants include variants in which a small portion(e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removedfrom the N- and/or C-terminal of the mature protein.

In many instances, a variant will contain conservative substitutions. A“conservative substitution” is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. As described above, modifications may be made in thestructure of the polynucleotides and polypeptides of the presentinvention and still obtain a functional molecule that encodes a variantor derivative polypeptide with desirable characteristics, e.g., withimmunogenic characteristics. When it is desired to alter the amino acidsequence of a polypeptide to create an equivalent, or even an improved,immunogenic variant or portion of a polypeptide of the invention, oneskilled in the art will typically change one or more of the codons ofthe encoding DNA sequence according to Table 1.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated that various changes may bemade in the peptide sequences of the disclosed compositions, orcorresponding DNA sequences which encode said peptides withoutappreciable loss of their biological utility or activity.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUPraline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporated herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of its hydrophobicity and charge characteristics(Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred. It is also understoodin the art that the substitution of like amino acids can be madeeffectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101(specifically incorporated herein by reference in its entirety), statesthat the greatest local average hydrophilicity of a protein, as governedby the hydrophilicity of its adjacent amino acids, correlates with abiological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions that take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

In addition, any polynucleotide may be further modified to increasestability in vivo. Possible modifications include, but are not limitedto, the addition of flanking sequences at the 5′ and/or 3′ ends; the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages in the backbone; and/or the inclusion of nontraditional basessuch as inosine, queosine and wybutosine, as well as acetyl- methyl-,thio- and other modified forms of adenine, cytidine, guanine, thymineand uridine.

Amino acid substitutions may further be made on the basis of similarityin polarity, charge, solubility, hydrophobicity, hydrophilicity and/orthe amphipathic nature of the residues. For example, negatively chargedamino acids include aspartic acid and glutamic acid; positively chargedamino acids include lysine and arginine; and amino acids with unchargedpolar head groups having similar hydrophilicity values include leucine,isoleucine and valine; glycine and alanine; asparagine and glutamine;and serine, threonine, phenylalanine and tyrosine. Other groups of aminoacids that may represent conservative changes include: (1) ala, pro,gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile,leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Avariant may also, or alternatively, contain nonconservative changes. Ina preferred embodiment, variant polypeptides differ from a nativesequence by substitution, deletion or addition of five amino acids orfewer. Variants may also (or alternatively) be modified by, for example,the deletion or addition of amino acids that have minimal influence onthe immunogenicity, secondary structure and hydropathic nature of thepolypeptide.

As noted above, polypeptides may comprise a signal (or leader) sequenceat the N-terminal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-His), or to enhance binding of the polypeptide to a solid support.For example, a polypeptide may be conjugated to an immunoglobulin Fcregion.

When comparing polypeptide sequences, two sequences are said to be“identical” if the sequence of amino acids in the two sequences is thesame when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Nat'l Acad., Sci. USA80:726-730.

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman (1981)Add. APL. Math 2:482, by the identity alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443, by the search for similaritymethods of Pearson and Lipman (1988) Proc. Nat'l Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or byinspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al. (1977)Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively. BLAST and BLAST 2.0 can be used, for examplewith the parameters described herein, to determine percent sequenceidentity for the polynucleotides and polypeptides of the invention.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. For amino acid sequences,a scoring matrix can be used to calculate the cumulative score.Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment.

In one preferred approach, the “percentage of sequence identity” isdetermined by comparing two optimally aligned sequences over a window ofcomparison of at least 20 positions, wherein the portion of thepolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the referencesequence (i.e., the window size) and multiplying the results by 100 toyield the percentage of sequence identity.

Within other illustrative embodiments, a polypeptide may be a fusionpolypeptide that comprises multiple polypeptides as described herein, orthat comprises at least one polypeptide as described herein and anunrelated sequence, such as a known tumor protein. A fusion partner may,for example, assist in providing T helper epitopes (an immunologicalfusion partner), preferably T helper epitopes recognized by humans, ormay assist in expressing the protein (an expression enhancer) at higheryields than the native recombinant protein. Certain preferred fusionpartners are both immunological and expression enhancing fusionpartners. Other fusion partners may be selected so as to increase thesolubility of the polypeptide or to enable the polypeptide to betargeted to desired intracellular compartments. Still further fusionpartners include affinity tags, which facilitate purification of thepolypeptide.

Fusion polypeptides may generally be prepared using standard techniques,including chemical conjugation. Preferably, a fusion polypeptide isexpressed as a recombinant polypeptide, allowing the production ofincreased levels, relative to a non-fused polypeptide, in an expressionsystem. Briefly, DNA sequences encoding the polypeptide components maybe assembled separately, and ligated into an appropriate expressionvector. The 3′ end of the DNA sequence encoding one polypeptidecomponent is ligated, with or without a peptide linker, to the 5′ end ofa DNA sequence encoding the second polypeptide component so that thereading frames of the sequences are in phase. This permits translationinto a single fusion polypeptide that retains the biological activity ofboth component polypeptides.

A peptide linker sequence may be employed to separate the first andsecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures. Sucha peptide linker sequence is incorporated into the fusion polypeptideusing standard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46, 1985; Murphy et al., Proc. Nat'l Acad. Sci. USA83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180.The linker sequence may generally be from 1 to about 50 amino acids inlength. Linker sequences are not required when the first and secondpolypeptides have non-essential N-terminal amino acid regions that canbe used to separate the functional domains and prevent stericinterference.

The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.

The fusion polypeptide can comprise a polypeptide as described hereintogether with an unrelated immunogenic protein, such as an immunogenicprotein capable of eliciting a recall response. Examples of suchproteins include tetanus, tuberculosis and hepatitis proteins (see, forexample, Stoute et al. New Engl. J. Med., 336:86-91, 1997).

In one preferred embodiment, the immunological fusion partner is derivedfrom a Mycobacterium sp., such as a Mycobacterium tuberculosis-derivedRa12 fragment. Ra12 compositions and methods for their use in enhancingthe expression and/or immunogenicity of heterologouspolynucleotide/polypeptide sequences is described in U.S. PatentApplication 60/158,585, the disclosure of which is incorporated hereinby reference in its entirety. Briefly, Ra12 refers to a polynucleotideregion that is a subsequence of a Mycobacterium tuberculosis MTB32Anucleic acid. MTB32A is a serine protease of 32 KD molecular weightencoded by a gene in virulent and avirulent strains of M. tuberculosis.The nucleotide sequence and amino acid sequence of MTB32A have beendescribed (for example, U.S. Patent Application 60/158,585; see also,Skeiky et al., Infection and Immun. (1999) 67:3998-4007, incorporatedherein by reference). C-terminal fragments of the MTB32A coding sequenceexpress at high levels and remain as a soluble polypeptides throughoutthe purification process. Moreover, Ra12 may enhance the immunogenicityof heterologous immunogenic polypeptides with which it is fused. Onepreferred Ra12 fusion polypeptide comprises a 14 KD C-terminal fragmentcorresponding to amino acid residues 192 to 323 of MTB32A. Otherpreferred Ra12 polynucleotides generally comprise at least about 15consecutive nucleotides, at least about 30 nucleotides, at least about60 nucleotides, at least about 100 nucleotides, at least about 200nucleotides, or at least about 300 nucleotides that encode a portion ofa Ra12 polypeptide. Ra12 polynucleotides may comprise a native sequence(i.e., an endogenous sequence that encodes a Ra12 polypeptide or aportion thereof) or may comprise a variant of such a sequence. Ra12polynucleotide variants may contain one or more substitutions,additions, deletions and/or insertions such that the biological activityof the encoded fusion polypeptide is not substantially diminished,relative to a fusion polypeptide comprising a native Ra12 polypeptide.Variants preferably exhibit at least about 70% identity, more preferablyat least about 80% identity and most preferably at least about 90%identity to a polynucleotide sequence that encodes a native Ra12polypeptide or a portion thereof.

Within other preferred embodiments, an immunological fusion partner isderived from protein D, a surface protein of the gram-negative bacteriumHaemophilus influenza B (WO 91/18926). Preferably, a protein Dderivative comprises approximately the first third of the protein (e.g.,the first N-terminal 100-110 amino acids), and a protein D derivativemay be lipidated. Within certain preferred embodiments, the first 109residues of a Lipoprotein D fusion partner is included on the N-terminusto provide the polypeptide with additional exogenous T-cell epitopes andto increase the expression level in E. coli (thus functioning as anexpression enhancer). The lipid tail ensures optimal presentation of theantigen to antigen presenting cells. Other fusion partners include thenon-structural protein from influenzae virus, NS1 (hemaglutinin).Typically, the N-terminal 81 amino acids are used, although differentfragments that include T-helper epitopes may be used.

In another embodiment, the immunological fusion partner is the proteinknown as LYTA, or a portion thereof (preferably a C-terminal portion).LYTA is derived from Streptococcus pneumoniae, which synthesizes anN-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytAgene; Gene 43:265-292, 1986). LYTA is an autolysin that specificallydegrades certain bonds in the peptidoglycan backbone. The C-terminaldomain of the LYTA protein is responsible for the affinity to thecholine or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798, 1992). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionpolypeptide. A repeat portion is found in the C-terminal region startingat residue 178. A particularly preferred repeat portion incorporatesresidues 188-305.

Yet another illustrative embodiment involves fusion polypeptides, andthe polynucleotides encoding them, wherein the fusion partner comprisesa targeting signal capable of directing a polypeptide to theendosomal/lysosomal compartment, as described in U.S. Pat. No.5,633,234. An immunogenic polypeptide of the invention, when fused withthis targeting signal, will associate more efficiently with MHC class IImolecules and thereby provide enhanced in vivo stimulation of CD4⁺T-cells specific for the polypeptide.

Polypeptides of the invention are prepared using any of a variety ofwell known synthetic and/or recombinant techniques, the latter of whichare further described below. Polypeptides, portions and other variantsgenerally less than about 150 amino acids can be generated by syntheticmeans, using techniques well known to those of ordinary skill in theart. In one illustrative example, such polypeptides are synthesizedusing any of the commercially available solid-phase techniques, such asthe Merrifield solid-phase synthesis method, where amino acids aresequentially added to a growing amino acid chain. See Merrifield, J. Am.Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis ofpolypeptides is commercially available from suppliers such as PerkinElmer/Applied BioSystems Division (Foster City, Calif.), and may beoperated according to the manufacturer's instructions.

In general, polypeptide compositions (including fusion polypeptides) ofthe invention are isolated. An “isolated” polypeptide is one that isremoved from its original environment. For example, anaturally-occurring protein or polypeptide is isolated if it isseparated from some or all of the coexisting materials in the naturalsystem. Preferably, such polypeptides are also purified, e.g., are atleast about 90% pure, more preferably at least about 95% pure and mostpreferably at least about 99% pure.

Polynucleotide Compositions

The present invention, in other aspects, provides polynucleotidecompositions. The terms “DNA” and “polynucleotide” are used essentiallyinterchangeably herein to refer to a DNA molecule that has been isolatedfree of total genomic DNA of a particular species. “Isolated,” as usedherein, means that a polynucleotide is substantially away from othercoding sequences, and that the DNA molecule does not contain largeportions of unrelated coding DNA, such as large chromosomal fragments orother functional genes or polypeptide coding regions. Of course, thisrefers to the DNA molecule as originally isolated, and does not excludegenes or coding regions later added to the segment by the hand of man.

As will be understood by those skilled in the art, the polynucleotidecompositions of this invention can include genomic sequences,extra-genomic and plasmid-encoded sequences and smaller engineered genesegments that express, or may be adapted to express, proteins,polypeptides, peptides and the like. Such segments may be naturallyisolated, or modified synthetically by the hand of man.

As will be also recognized by the skilled artisan, polynucleotides ofthe invention may be single-stranded (coding or antisense) ordouble-stranded, and may be DNA (genomic, cDNA or synthetic) or RNAmolecules. RNA molecules may include HnRNA molecules, which containintrons and correspond to a DNA molecule in a one-to-one manner, andmRNA molecules, which do not contain introns. Additional coding ornon-coding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a polypeptide/protein of the invention or aportion thereof) or may comprise a sequence that encodes a variant orderivative, preferably an immunogenic variant or derivative, of such asequence.

Therefore, according to another aspect of the present invention,polynucleotide compositions are provided that comprise some or all of apolynucleotide sequence set forth in any one of SEQ ID NO: 1-111,115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335,340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552,569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824,878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939, 942, 944,945, 948, 967, 969-976, 991, 993-1002, 1004, 1006, 1007, 1010,1013-1019, 1021, 1023-1027 and 1034-1036, complements of apolynucleotide sequence set forth in any one of SEQ ID NO: 1-111,115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335,340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552,569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824,878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939, 942, 944,945, 948, 967, 969-976, 991, 993-1002, 1004, 1006, 1007, 1010,1013-1019, 1021, 1023-1027 and 1034-1036, and degenerate variants of apolynucleotide sequence set forth in any one of SEQ ID NO: 1-111,115-171, 173-175, 177, 179-305, 307-315, 326, 328, 330, 332-335,340-375, 381, 382 and 384-476, 524, 526, 530, 531, 533, 535, 536, 552,569-572, 587, 591, 593-606, 618-705, 709-774, 777, 789, 817, 823, 824,878, 880-882, 894, 896, 907, 908, 916-919, 929-931, 938, 939, 942, 944,945, 948, 967, 969-976, 991, 993-1002, 1004, 1006, 1007, 1010,1013-1019, 1021, 1023-1027 and 1034-1036. In certain preferredembodiments, the polynucleotide sequences set forth herein encodeimmunogenic polypeptides, as described above.

In other related embodiments, the present invention providespolynucleotide variants having substantial identity to the sequencesdisclosed herein in SEQ ID NO: 1-111, 115-171, 173-175, 177, 179-305,307-315, 326, 328, 330, 332-335, 340-375, 381, 382 and 384-476, 524,526, 530, 531, 533, 535, 536, 552, 569-572, 587, 591, 593-606, 618-705,709-774, 777, 789, 817, 823, 824, 878, 880-882, 894, 896, 907, 908,916-919, 929-931, 938, 939, 942, 944, 945, 948, 967, 969-976, 991,993-1002, 1004, 1006, 1007, 1010, 1013-1019, 1021, 1023-1027 and1034-1036 for example those comprising at least 70% sequence identity,preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% orhigher, sequence identity compared to a polynucleotide sequence of thisinvention using the methods described herein, (e.g., BLAST analysisusing standard parameters, as described below). One skilled in this artwill recognize that these values can be appropriately adjusted todetermine corresponding identity of proteins encoded by two nucleotidesequences by taking into account codon degeneracy, amino acidsimilarity, reading frame positioning and the like.

Typically, polynucleotide variants will contain one or moresubstitutions, additions, deletions and/or insertions, preferably suchthat the immunogenicity of the polypeptide encoded by the variantpolynucleotide is not substantially diminished relative to a polypeptideencoded by a polynucleotide sequence specifically set forth herein). Theterm “variants” should also be understood to encompasses homologousgenes of xenogenic origin.

In additional embodiments, the present invention provides polynucleotidefragments comprising various lengths of contiguous stretches of sequenceidentical to, or complementary to, one or more of the sequencesdisclosed herein. For example, polynucleotides are provided by thisinvention that comprise at least about 10, 15, 20, 30, 40, 50, 75, 100,150, 200, 300, 400, 500 or 1000 or more contiguous nucleotides of one ormore of the sequences disclosed herein as well as all intermediatelengths there between. It will be readily understood that “intermediatelengths”, in this context, means any length between the quoted values,such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50,51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.;including all integers through 200-500; 500-1,000, and the like.

In another embodiment of the invention, polynucleotide compositions areprovided that are capable of hybridizing under moderate to highstringency conditions to a polynucleotide sequence provided herein, or afragment thereof, or a complementary sequence thereof. Hybridizationtechniques are well known in the art of molecular biology. For purposesof illustration, suitable moderately stringent conditions for testingthe hybridization of a polynucleotide of this invention with otherpolynucleotides include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0mM EDTA (pH 8.0); hybridizing at 50° C.-60° C., 5×SSC, overnight;followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5×and 0.2×SSC containing 0.1% SDS. One skilled in the art will understandthat the stringency of hybridization can be readily manipulated, such asby altering the salt content of the hybridization solution and/or thetemperature at which the hybridization is performed. For example, inanother embodiment, suitable highly stringent hybridization conditionsinclude those described above, with the exception that the temperatureof hybridization is increased, e.g., to 60-65° C. or 65-70° C.

In certain preferred embodiments, the polynucleotides described above,e.g., polynucleotide variants, fragments and hybridizing sequences,encode polypeptides that are immunologically cross-reactive with apolypeptide sequence specifically set forth herein. In other preferredembodiments, such polynucleotides encode polypeptides that have a levelof immunogenic activity of at least about 50%, preferably at least about70%, and more preferably at least about 90% of that for a polypeptidesequence specifically set forth herein.

The polynucleotides of the present invention, or fragments thereof,regardless of the length of the coding sequence itself, may be combinedwith other DNA sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol. For example, illustrative polynucleotidesegments with total lengths of about 10,000, about 5000, about 3000,about 2,000, about 1,000, about 500, about 200, about 100, about 50 basepairs in length, and the like, (including all intermediate lengths) arecontemplated to be useful in many implementations of this invention.

When comparing polynucleotide sequences, two sequences are said to be“identical” if the sequence of nucleotides in the two sequences is thesame when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, preferably 40 to about 50, in which a sequence may becompared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Nat'l Acad., Sci. USA80:726-730.

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman (1981)Add. APL. Math 2:482, by the identity alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443, by the search for similaritymethods of Pearson and Lipman (1988) Proc. Nat'l Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or byinspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al. (1977)Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively. BLAST and BLAST 2.0 can be used, for examplewith the parameters described herein, to determine percent sequenceidentity for the polynucleotides of the invention. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. In one illustrative example,cumulative scores can be calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Nat'l Acad. Sci. USA 89:10915)alignments, (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparisonof both strands.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12percent, as compared to the reference sequences (which does not compriseadditions or deletions) for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whichthe identical nucleic acid bases occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the reference sequence (i.e., thewindow size) and multiplying the results by 100 to yield the percentageof sequence identity.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Nonetheless, polynucleotides that vary due to differencesin codon usage are specifically contemplated by the present invention.Further, alleles of the genes comprising the polynucleotide sequencesprovided herein are within the scope of the present invention. Allelesare endogenous genes that are altered as a result of one or moremutations, such as deletions, additions and/or substitutions ofnucleotides. The resulting mRNA and protein may, but need not, have analtered structure or function. Alleles may be identified using standardtechniques (such as hybridization, amplification and/or databasesequence comparison).

Therefore, in another embodiment of the invention, a mutagenesisapproach, such as site-specific mutagenesis, is employed for thepreparation of immunogenic variants and/or derivatives of thepolypeptides described herein. By this approach, specific modificationsin a polypeptide sequence can be made through mutagenesis of theunderlying polynucleotides that encode them. These techniques provides astraightforward approach to prepare and test sequence variants, forexample, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into thepolynucleotide.

Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Mutations may be employed in aselected polynucleotide sequence to improve, alter, decrease, modify, orotherwise change the properties of the polynucleotide itself, and/oralter the properties, activity, composition, stability, or primarysequence of the encoded polypeptide.

In certain embodiments of the present invention, the inventorscontemplate the mutagenesis of the disclosed polynucleotide sequences toalter one or more properties of the encoded polypeptide, such as theimmunogenicity of a polypeptide vaccine. The techniques of site-specificmutagenesis are well-known in the art, and are widely used to createvariants of both polypeptides and polynucleotides. For example,site-specific mutagenesis is often used to alter a specific portion of aDNA molecule. In such embodiments, a primer comprising typically about14 to about 25 nucleotides or so in length is employed, with about 5 toabout 10 residues on both sides of the junction of the sequence beingaltered.

As will be appreciated by those of skill in the art, site-specificmutagenesis techniques have often employed a phage vector that exists inboth a single stranded and double stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage are readily commercially-available and their use isgenerally well-known to those skilled in the art. Double-strandedplasmids are also routinely employed in site directed mutagenesis thateliminates the step of transferring the gene of interest from a plasmidto a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double-stranded vector that includes within itssequence a DNA sequence that encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis provides a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.Specific details regarding these methods and protocols are found in theteachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991;Kuby, 1994; and Maniatis et al., 1982, each incorporated herein byreference, for that purpose.

As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” is intended to refer to a process that involvesthe template-dependent extension of a primer molecule. The term templatedependent process refers to nucleic acid synthesis of an RNA or a DNAmolecule wherein the sequence of the newly synthesized strand of nucleicacid is dictated by the well-known rules of complementary base pairing(see, for example, Watson, 1987). Typically, vector mediatedmethodologies involve the introduction of the nucleic acid fragment intoa DNA or RNA vector, the clonal amplification of the vector, and therecovery of the amplified nucleic acid fragment. Examples of suchmethodologies are provided by U.S. Pat. No. 4,237,224, specificallyincorporated herein by reference in its entirety.

In another approach for the production of polypeptide variants of thepresent invention, recursive sequence recombination, as described inU.S. Pat. No. 5,837,458, may be employed. In this approach, iterativecycles of recombination and screening or selection are performed to“evolve” individual polynucleotide variants of the invention having, forexample, enhanced immunogenic activity.

In other embodiments of the present invention, the polynucleotidesequences provided herein can be advantageously used as probes orprimers for nucleic acid hybridization. As such, it is contemplated thatnucleic acid segments that comprise a sequence region of at least about15 contiguous nucleotides that has the same sequence as, or iscomplementary to, a 15 nucleotide long contiguous sequence disclosedherein will find particular utility. Longer contiguous identical orcomplementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200,500, 1000 (including all intermediate lengths) and even up to fulllength sequences will also be of use in certain embodiments.

The ability of such nucleic acid probes to specifically hybridize to asequence of interest will enable them to be of use in detecting thepresence of complementary sequences in a given sample. However, otheruses are also envisioned, such as the use of the sequence informationfor the preparation of mutant species primers, or primers for use inpreparing other genetic constructions.

Polynucleotide molecules having sequence regions consisting ofcontiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of100-200 nucleotides or so (including intermediate lengths as well),identical or complementary to a polynucleotide sequence disclosedherein, are particularly contemplated as hybridization probes for usein, e.g., Southern and Northern blotting. This would allow a geneproduct, or fragment thereof, to be analyzed, both in diverse cell typesand also in various bacterial cells. The total size of fragment, as wellas the size of the complementary stretch(es), will ultimately depend onthe intended use or application of the particular nucleic acid segment.Smaller fragments will generally find use in hybridization embodiments,wherein the length of the contiguous complementary region may be varied,such as between about 15 and about 100 nucleotides, but largercontiguous complementarity stretches may be used, according to thelength complementary sequences one wishes to detect.

The use of a hybridization probe of about 15-25 nucleotides in lengthallows the formation of a duplex molecule that is both stable andselective. Molecules having contiguous complementary sequences overstretches greater than 15 bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained. One will generally prefer to design nucleic acid moleculeshaving gene-complementary stretches of 15 to 25 contiguous nucleotides,or even longer where desired.

Hybridization probes may be selected from any portion of any of thesequences disclosed herein. All that is required is to review thesequences set forth herein, or to any continuous portion of thesequences, from about 15-25 nucleotides in length up to and includingthe full length sequence, that one wishes to utilize as a probe orprimer. The choice of probe and primer sequences may be governed byvarious factors. For example, one may wish to employ primers fromtowards the termini of the total sequence.

Small polynucleotide segments or fragments may be readily prepared by,for example, directly synthesizing the fragment by chemical means, as iscommonly practiced using an automated oligonucleotide synthesizer. Also,fragments may be obtained by application of nucleic acid reproductiontechnology, such as the PCR™ technology of U.S. Pat. No. 4,683,202(incorporated herein by reference), by introducing selected sequencesinto recombinant vectors for recombinant production, and by otherrecombinant DNA techniques generally known to those of skill in the artof molecular biology.

The nucleotide sequences of the invention may be used for their abilityto selectively form duplex molecules with complementary stretches of theentire gene or gene fragments of interest. Depending on the applicationenvisioned, one will typically desire to employ varying conditions ofhybridization to achieve varying degrees of selectivity of probe towardstarget sequence. For applications requiring high selectivity, one willtypically desire to employ relatively stringent conditions to form thehybrids, e.g., one will select relatively low salt and/or hightemperature conditions, such as provided by a salt concentration of fromabout 0.02 M to about 0.15 M salt at temperatures of from about 50° C.to about 70° C. Such selective conditions tolerate little, if any,mismatch between the probe and the template or target strand, and wouldbe particularly suitable for isolating related sequences.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template, less stringent (reduced stringency) hybridizationconditions will typically be needed in order to allow formation of theheteroduplex. In these circumstances, one may desire to employ saltconditions such as those of from about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Cross-hybridizingspecies can thereby be readily identified as positively hybridizingsignals with respect to control hybridizations. In any case, it isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide, which serves todestabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

According to another embodiment of the present invention, polynucleotidecompositions comprising antisense oligonucleotides are provided.Antisense oligonucleotides have been demonstrated to be effective andtargeted inhibitors of protein synthesis, and, consequently, provide atherapeutic approach by which a disease can be treated by inhibiting thesynthesis of proteins that contribute to the disease. The efficacy ofantisense oligonucleotides for inhibiting protein synthesis is wellestablished. For example, the synthesis of polygalactauronase and themuscarine type 2 acetylcholine receptor are inhibited by antisenseoligonucleotides directed to their respective mRNA sequences (U.S. Pat.No. 5,739,119 and U.S. Pat. No. 5,759,829). Further, examples ofantisense inhibition have been demonstrated with the nuclear proteincyclin, the multiple drug resistance gene (MDG1), ICAM-1, E-selectin,STK-1, striatal GABA_(A) receptor and human EGF (Jaskulski et al.,Science. 1988 Jun. 10; 240(4858):1544-6; Vasanthakumar and Ahmed, CancerCommun. 1989; 11(4):225-32; Peris et al., Brain Res Mol Brain Res. 1998Jun. 15; 57(2):310-20; U.S. Pat. No. 5,801,154; U.S. Pat. No. 5,789,573;U.S. Pat. No. 5,718,709 and U.S. Pat. No. 5,610,288). Antisenseconstructs have also been described that inhibit and can be used totreat a variety of abnormal cellular proliferations, e.g. cancer (U.S.Pat. No. 5,747,470; U.S. Pat. No. 5,591,317 and U.S. Pat. No.5,783,683).

Therefore, in certain embodiments, the present invention providesoligonucleotide sequences that comprise all, or a portion of, anysequence that is capable of specifically binding to polynucleotidesequence described herein, or a complement thereof. In one embodiment,the antisense oligonucleotides comprise DNA or derivatives thereof. Inanother embodiment, the oligonucleotides comprise RNA or derivativesthereof. In a third embodiment, the oligonucleotides are modified DNAscomprising a phosphorothioated modified backbone. In a fourthembodiment, the oligonucleotide sequences comprise peptide nucleic acidsor derivatives thereof. In each case, preferred compositions comprise asequence region that is complementary, and more preferablysubstantially-complementary, and even more preferably, completelycomplementary to one or more portions of polynucleotides disclosedherein. Selection of antisense compositions specific for a given genesequence is based upon analysis of the chosen target sequence anddetermination of secondary structure, T_(m), binding energy, andrelative stability. Antisense compositions may be selected based upontheir relative inability to form dimers, hairpins, or other secondarystructures that would reduce or prohibit specific binding to the targetmRNA in a host cell. Highly preferred target regions of the mRNA, arethose which are at or near the AUG translation initiation codon, andthose sequences which are substantially complementary to 5′ regions ofthe mRNA. These secondary structure analyses and target site selectionconsiderations can be performed, for example, using v.4 of the OLIGOprimer analysis software and/or the BLASTN 2.0.5 algorithm software(Altschul et al., Nucleic Acids Res. 1997 Sep. 1; 25(17):3389-402).

The use of an antisense delivery method employing a short peptidevector, termed MPG (27 residues), is also contemplated. The MPG peptidecontains a hydrophobic domain derived from the fusion sequence of HIVgp41 and a hydrophilic domain from the nuclear localization sequence ofSV40 T-antigen (Morris et al., Nucleic Acids Res. 1997 Jul. 15;25(14):2730-6). It has been demonstrated that several molecules of theMPG peptide coat the antisense oligonucleotides and can be deliveredinto cultured mammalian cells in less than 1 hour with relatively highefficiency (90%). Further, the interaction with MPG strongly increasesboth the stability of the oligonucleotide to nuclease and the ability tocross the plasma membrane.

According to another embodiment of the invention, the polynucleotidecompositions described herein are used in the design and preparation ofribozyme molecules for inhibiting expression of the tumor polypeptidesand proteins of the present invention in tumor cells. Ribozymes areRNA-protein complexes that cleave nucleic acids in a site-specificfashion. Ribozymes have specific catalytic domains that possessendonuclease activity (Kim and Cech, Proc Nat'l Acad Sci U S A. 1987December; 84(24):8788-92; Forster and Symons, Cell. 1987 Apr. 24;49(2):211-20). For example, a large number of ribozymes acceleratephosphoester transfer reactions with a high degree of specificity, oftencleaving only one of several phosphoesters in an oligonucleotidesubstrate (Cech et al., Cell. 1981 December; 27(3 Pt 2):487-96; Micheland Westhof, J Mol Biol. 1990 Dec. 5; 216(3):585-610; Reinhold-Hurek andShub, Nature. 1992 May 14; 357(6374):173-6). This specificity has beenattributed to the requirement that the substrate bind via specificbase-pairing interactions to the internal guide sequence (“IGS”) of theribozyme prior to chemical reaction.

Six basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans (and thus can cleave other RNA molecules) under physiologicalconditions. In general, enzymatic nucleic acids act by first binding toa target RNA. Such binding occurs through the target binding portion ofa enzymatic nucleic acid which is held in close proximity to anenzymatic portion of the molecule that acts to cleave the target RNA.Thus, the enzymatic nucleic acid first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic nucleic acid hasbound and cleaved its RNA target, it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over manytechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the concentration of ribozyme necessary to affect a therapeutictreatment is lower than that of an antisense oligonucleotide. Thisadvantage reflects the ability of the ribozyme to act enzymatically.Thus, a single ribozyme molecule is able to cleave many molecules oftarget RNA. In addition, the ribozyme is a highly specific inhibitor,with the specificity of inhibition depending not only on the basepairing mechanism of binding to the target RNA, but also on themechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of a ribozyme. Similar mismatches in antisensemolecules do not prevent their action (Woolf et al., Proc Nat'l Acad SciUSA. 1992 Aug. 15; 89(16):7305-9). Thus, the specificity of action of aribozyme is greater than that of an antisense oligonucleotide bindingthe same RNA site.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, a hepatitis δ virus, group I intron or RNaseP RNA (inassociation with an RNA guide sequence) or Neurospora VS RNA motif.Examples of hammerhead motifs are described by Rossi et al. NucleicAcids Res. 1992 Sep. 11; 20(17):4559-65. Examples of hairpin motifs aredescribed by Hampel et al. (Eur. Pat. Appl. Publ. No. EP 0360257),Hampel and Tritz, Biochemistry 1989 Jun. 13; 28(12):4929-33; Hampel etal., Nucleic Acids Res. 1990 Jan. 25; 18(2):299-304 and U.S. Pat. No.5,631,359. An example of the hepatitis δ virus motif is described byPerrotta and Been, Biochemistry. 1992 Dec. 1; 31(47):11843-52; anexample of the RNaseP motif is described by Guerrier-Takada et al.,Cell. 1983 December; 35(3 Pt 2):849-57; Neurospora VS RNA ribozyme motifis described by Collins (Saville and Collins, Cell. 1990 May 18;61(4):685-96; Saville and Collins, Proc Nat'l Acad Sci USA. 1991 Oct. 1;88(19):8826-30; Collins and Olive, Biochemistry. 1993 Mar. 23;32(11):2795-9); and an example of the Group I intron is described in(U.S. Pat. No. 4,987,071). All that is important in an enzymatic nucleicacid molecule of this invention is that it has a specific substratebinding site which is complementary to one or more of the target geneRNA regions, and that it have nucleotide sequences within or surroundingthat substrate binding site which impart an RNA cleaving activity to themolecule. Thus the ribozyme constructs need not be limited to specificmotifs mentioned herein.

Ribozymes may be designed as described in Int. Pat. Appl. Publ. No. WO93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595, each specificallyincorporated herein by reference) and synthesized to be tested in vitroand in vivo, as described. Such ribozymes can also be optimized fordelivery. While specific examples are provided, those in the art willrecognize that equivalent RNA targets in other species can be utilizedwhen necessary.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms, or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g., Int. Pat. Appl. Publ. No. WO 92/07065; Int. Pat. Appl. Publ. No.WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl.Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ.No. WO 94/13688, which describe various chemical modifications that canbe made to the sugar moieties of enzymatic RNA molecules), modificationswhich enhance their efficacy in cells, and removal of stem II bases toshorten RNA synthesis times and reduce chemical requirements.

Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describes thegeneral methods for delivery of enzymatic RNA molecules. Ribozymes maybe administered to cells by a variety of methods known to those familiarto the art, including, but not restricted to, encapsulation inliposomes, by iontophoresis, or by incorporation into other vehicles,such as hydrogels, cyclodextrins, biodegradable nanocapsules, andbioadhesive microspheres. For some indications, ribozymes may bedirectly delivered ex vivo to cells or tissues with or without theaforementioned vehicles. Alternatively, the RNA/vehicle combination maybe locally delivered by direct inhalation, by direct injection or by useof a catheter, infusion pump or stent. Other routes of delivery include,but are not limited to, intravascular, intramuscular, subcutaneous orjoint injection, aerosol inhalation, oral (tablet or pill form),topical, systemic, ocular, intraperitoneal and/or intrathecal delivery.More detailed descriptions of ribozyme delivery and administration areprovided in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl.Publ. No. WO 93/23569, each specifically incorporated herein byreference.

Another means of accumulating high concentrations of a ribozyme(s)within cells is to incorporate the ribozyme-encoding sequences into aDNA expression vector. Transcription of the ribozyme sequences aredriven from a promoter for eukaryotic RNA polymerase I (pol I), RNApolymerase II (pol II), or RNA polymerase III (pol III). Transcriptsfrom pol II or pol III promoters will be expressed at high levels in allcells; the levels of a given pol II promoter in a given cell type willdepend on the nature of the gene regulatory sequences (enhancers,silencers, etc.) present nearby. Prokaryotic RNA polymerase promotersmay also be used, providing that the prokaryotic RNA polymerase enzymeis expressed in the appropriate cells Ribozymes expressed from suchpromoters have been shown to function in mammalian cells. Suchtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated vectors), or viral RNA vectors (such as retroviral,semliki forest virus, sindbis virus vectors).

In another embodiment of the invention, peptide nucleic acids (PNAs)compositions are provided. PNA is a DNA mimic in which the nucleobasesare attached to a pseudopeptide backbone (Good and Nielsen, AntisenseNucleic Acid Drug Dev. 1997 7(4) 431-37). PNA is able to be utilized ina number methods that traditionally have used RNA or DNA. Often PNAsequences perform better in techniques than the corresponding RNA or DNAsequences and have utilities that are not inherent to RNA or DNA. Areview of PNA including methods of making, characteristics of, andmethods of using, is provided by Corey (Trends Biotechnol 1997 June;15(6):224-9). As such, in certain embodiments, one may prepare PNAsequences that are complementary to one or more portions of the ACE mRNAsequence, and such PNA compositions may be used to regulate, alter,decrease, or reduce the translation of ACE-specific mRNA, and therebyalter the level of ACE activity in a host cell to which such PNAcompositions have been administered.

PNAs have 2-aminoethyl-glycine linkages replacing the normalphosphodiester backbone of DNA (Nielsen et al., Science 1991 Dec. 6;254(5037):1497-500; Hanvey et al., Science. 1992 Nov. 27;258(5087):1481-5; Hyrup and Nielsen, Bioorg Med. Chem. 1996 January;4(1):5-23). This chemistry has three important consequences: firstly, incontrast to DNA or phosphorothioate oligonucleotides, PNAs are neutralmolecules; secondly, PNAs are achiral, which avoids the need to developa stereoselective synthesis; and thirdly, PNA synthesis uses standardBoc or Fmoc protocols for solid-phase peptide synthesis, although othermethods, including a modified Merrifield method, have been used.

PNA monomers or ready-made oligomers are commercially available fromPerSeptive Biosystems (Framingham, Mass.). PNA syntheses by either Bocor Fmoc protocols are straightforward using manual or automatedprotocols (Norton et al., Bioorg Med Chem. 1995 April; 3(4):437-45). Themanual protocol lends itself to the production of chemically modifiedPNAs or the simultaneous synthesis of families of closely related PNAs.

As with peptide synthesis, the success of a particular PNA synthesiswill depend on the properties of the chosen sequence. For example, whilein theory PNAs can incorporate any combination of nucleotide bases, thepresence of adjacent purines can lead to deletions of one or moreresidues in the product. In expectation of this difficulty, it issuggested that, in producing PNAs with adjacent purines, one shouldrepeat the coupling of residues likely to be added inefficiently. Thisshould be followed by the purification of PNAs by reverse-phasehigh-pressure liquid chromatography, providing yields and purity ofproduct similar to those observed during the synthesis of peptides.

Modifications of PNAs for a given application may be accomplished bycoupling amino acids during solid-phase synthesis or by attachingcompounds that contain a carboxylic acid group to the exposed N-terminalamine. Alternatively, PNAs can be modified after synthesis by couplingto an introduced lysine or cysteine. The ease with which PNAs can bemodified facilitates optimization for better solubility or for specificfunctional requirements. Once synthesized, the identity of PNAs andtheir derivatives can be confirmed by mass spectrometry. Several studieshave made and utilized modifications of PNAs (for example, Norton etal., Bioorg Med Chem. 1995 April; 3(4):437-45; Petersen et al., J PeptSci. 1995 May-June; 1(3):175-83; Orum et al., Biotechniques. 1995September; 19(3):472-80; Footer et al., Biochemistry. 1996 Aug. 20;35(33):10673-9; Griffith et al., Nucleic Acids Res. 1995 Aug. 11;23(15):3003-8; Pardridge et al., Proc Nat'l Acad Sci USA. 1995 Jun. 6;92(12):5592-6; Boffa et al., Proc Nat'l Acad Sci USA. 1995 Mar. 14;92(6):1901-5; Gambacorti-Passerini et al., Blood. 1996 Aug. 15;88(4):1411-7; Armitage et al., Proc Nat'l Acad Sci USA. 1997 Nov. 11;94(23):12320-5; Seeger et al., Biotechniques. 1997 September;23(3):512-7). U.S. Pat. No. 5,700,922 discusses PNA-DNA-PNA chimericmolecules and their uses in diagnostics, modulating protein inorganisms, and treatment of conditions susceptible to therapeutics.

Methods of characterizing the antisense binding properties of PNAs arediscussed in Rose (Anal Chem. 1993 Dec. 15; 65(24):3545-9) and Jensen etal. (Biochemistry. 1997 Apr. 22; 36(16):5072-7). Rose uses capillary gelelectrophoresis to determine binding of PNAs to their complementaryoligonucleotide, measuring the relative binding kinetics andstoichiometry. Similar types of measurements were made by Jensen et al.using BIAcore™ technology.

Other applications of PNAs that have been described and will be apparentto the skilled artisan include use in DNA strand invasion, antisenseinhibition, mutational analysis, enhancers of transcription, nucleicacid purification, isolation of transcriptionally active genes, blockingof transcription factor binding, genome cleavage, biosensors, in situhybridization, and the like.

Polynucleotide Identification, Characterization and Expression

Polynucleotide compositions of the present invention may be identified,prepared and/or manipulated using any of a variety of well establishedtechniques (see generally, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor,N.Y., 1989, and other like references). For example, a polynucleotidemay be identified, as described in more detail below, by screening amicroarray of cDNAs for tumor-associated expression (i.e., expressionthat is at least two fold greater in a tumor than in normal tissue, asdetermined using a representative assay provided herein). Such screensmay be performed, for example, using the microarray technology ofAffymetrix, Inc. (Santa Clara, Calif.) according to the manufacturer'sinstructions (and essentially as described by Schena et al., Proc. Nat'lAcad. Sci. USA 93:10614-10619, 1996 and Heller et al., Proc. Nat'l Acad.Sci. USA 94:2150-2155, 1997). Alternatively, polynucleotides may beamplified from cDNA prepared from cells expressing the proteinsdescribed herein, such as tumor cells.

Many template dependent processes are available to amplify a targetsequences of interest present in a sample. One of the best knownamplification methods is the polymerase chain reaction (PCR™) which isdescribed in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159, each of which is incorporated herein by reference in itsentirety. Briefly, in PCR™, two primer sequences are prepared which arecomplementary to regions on opposite complementary strands of the targetsequence. An excess of deoxynucleoside triphosphates is added to areaction mixture along with a DNA polymerase (e.g., Taq polymerase). Ifthe target sequence is present in a sample, the primers will bind to thetarget and the polymerase will cause the primers to be extended alongthe target sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the target to form reaction products, excess primerswill bind to the target and to the reaction product and the process isrepeated. Preferably reverse transcription and PCR™ amplificationprocedure may be performed in order to quantify the amount of mRNAamplified. Polymerase chain reaction methodologies are well known in theart.

Any of a number of other template dependent processes, many of which arevariations of the PCR™ amplification technique, are readily known andavailable in the art. Illustratively, some such methods include theligase chain reaction (referred to as LCR), described, for example, inEur. Pat. Appl. Publ. No. 320,308 and U.S. Pat. No. 4,883,750; QbetaReplicase, described in PCT Intl. Pat. Appl. Publ. No. PCT/US87/00880;Strand Displacement Amplification (SDA) and Repair Chain Reaction (RCR).Still other amplification methods are described in Great Britain Pat.Appl. No. 2 202 328, and in PCT Intl. Pat. Appl. Publ. No.PCT/US89/01025. Other nucleic acid amplification procedures includetranscription-based amplification systems (TAS) (PCT Intl. Pat. Appl.Publ. No. WO 88/10315), including nucleic acid sequence basedamplification (NASBA) and 3SR. Eur. Pat. Appl. Publ. No. 329,822describes a nucleic acid amplification process involving cyclicallysynthesizing single-stranded RNA (“ssRNA”), ssDNA, and double-strandedDNA (dsDNA). PCT Intl. Pat. Appl. Publ. No. WO 89/06700 describes anucleic acid sequence amplification scheme based on the hybridization ofa promoter/primer sequence to a target single-stranded DNA (“ssDNA”)followed by transcription of many RNA copies of the sequence. Otheramplification methods such as “RACE” (Frohman, 1990), and “one-sidedPCR” (Ohara, 1989) are also well-known to those of skill in the art.

An amplified portion of a polynucleotide of the present invention may beused to isolate a full length gene from a suitable library (e.g., atumor cDNA library) using well known techniques. Within such techniques,a library (cDNA or genomic) is screened using one or more polynucleotideprobes or primers suitable for amplification. Preferably, a library issize-selected to include larger molecules. Random primed libraries mayalso be preferred for identifying 5′ and upstream regions of genes.Genomic libraries are preferred for obtaining introns and extending 5′sequences.

For hybridization techniques, a partial sequence may be labeled (e.g.,by nick-translation or end-labeling with ³²P) using well knowntechniques. A bacterial or bacteriophage library is then generallyscreened by hybridizing filters containing denatured bacterial colonies(or lawns containing phage plaques) with the labeled probe (see Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y., 1989). Hybridizing colonies orplaques are selected and expanded, and the DNA is isolated for furtheranalysis. cDNA clones may be analyzed to determine the amount ofadditional sequence by, for example, PCR using a primer from the partialsequence and a primer from the vector. Restriction maps and partialsequences may be generated to identify one or more overlapping clones.The complete sequence may then be determined using standard techniques,which may involve generating a series of deletion clones. The resultingoverlapping sequences can then assembled into a single contiguoussequence. A full length cDNA molecule can be generated by ligatingsuitable fragments, using well known techniques.

Alternatively, amplification techniques, such as those described above,can be useful for obtaining a full length coding sequence from a partialcDNA sequence. One such amplification technique is inverse PCR (seeTriglia et al., Nucl. Acids Res. 16:8186, 1988), which uses restrictionenzymes to generate a fragment in the known region of the gene. Thefragment is then circularized by intramolecular ligation and used as atemplate for PCR with divergent primers derived from the known region.Within an alternative approach, sequences adjacent to a partial sequencemay be retrieved by amplification with a primer to a linker sequence anda primer specific to a known region. The amplified sequences aretypically subjected to a second round of amplification with the samelinker primer and a second primer specific to the known region. Avariation on this procedure, which employs two primers that initiateextension in opposite directions from the known sequence, is describedin WO 96/38591. Another such technique is known as “rapid amplificationof cDNA ends” or RACE. This technique involves the use of an internalprimer and an external primer, which hybridizes to a polyA region orvector sequence, to identify sequences that are 5′ and 3′ of a knownsequence. Additional techniques include capture PCR (Lagerstrom et al.,PCR Methods Applic. 1:111-19, 1991) and walking PCR (Parker et al.,Nucl. Acids. Res. 19:3055-60, 1991). Other methods employingamplification may also be employed to obtain a full length cDNAsequence.

In certain instances, it is possible to obtain a full length cDNAsequence by analysis of sequences provided in an expressed sequence tag(EST) database, such as that available from GenBank. Searches foroverlapping ESTs may generally be performed using well known programs(e.g., NCBI BLAST searches), and such ESTs may be used to generate acontiguous full length sequence. Full length DNA sequences may also beobtained by analysis of genomic fragments.

In other embodiments of the invention, polynucleotide sequences orfragments thereof which encode polypeptides of the invention, or fusionproteins or functional equivalents thereof, may be used in recombinantDNA molecules to direct expression of a polypeptide in appropriate hostcells. Due to the inherent degeneracy of the genetic code, other DNAsequences that encode substantially the same or a functionallyequivalent amino acid sequence may be produced and these sequences maybe used to clone and express a given polypeptide.

As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence.

Moreover, the polynucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterpolypeptide encoding sequences for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing, and/orexpression of the gene product. For example, DNA shuffling by randomfragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides may be used to engineer the nucleotide sequences. Inaddition, site-directed mutagenesis may be used to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, or introduce mutations, and soforth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences may be ligated to a heterologoussequence to encode a fusion protein. For example, to screen peptidelibraries for inhibitors of polypeptide activity, it may be useful toencode a chimeric protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the polypeptide-encoding sequence and theheterologous protein sequence, so that the polypeptide may be cleavedand purified away from the heterologous moiety.

Sequences encoding a desired polypeptide may be synthesized, in whole orin part, using chemical methods well known in the art (see Caruthers, M.H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al.(1980) Nucl. Acids Res. Symp. Ser. 225-232). Alternatively, the proteinitself may be produced using chemical methods to synthesize the aminoacid sequence of a polypeptide, or a portion thereof. For example,peptide synthesis can be performed using various solid-phase techniques(Roberge, J. Y. et al. (1995) Science 269:202-204) and automatedsynthesis may be achieved, for example, using the ABI 431A PeptideSynthesizer (Perkin Elmer, Palo Alto, Calif.).

A newly synthesized peptide may be substantially purified by preparativehigh performance liquid chromatography (e.g., Creighton, T. (1983)Proteins, Structures and Molecular Principles, WH Freeman and Co., NewYork, N.Y.) or other comparable techniques available in the art. Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure).Additionally, the amino acid sequence of a polypeptide, or any partthereof, may be altered during direct synthesis and/or combined usingchemical methods with sequences from other proteins, or any partthereof, to produce a variant polypeptide.

In order to express a desired polypeptide, the nucleotide sequencesencoding the polypeptide, or functional equivalents, may be insertedinto appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described, for example, in Sambrook,J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) CurrentProtocols in Molecular Biology, John Wiley & Sons, New York. N.Y.

A variety of expression vector/host systems may be utilized to containand express polynucleotide sequences. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thePBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

In bacterial systems, any of a number of expression vectors may beselected depending upon the use intended for the expressed polypeptide.For example, when large quantities are needed, for example for theinduction of antibodies, vectors which direct high level expression offusion proteins that are readily purified may be used. Such vectorsinclude, but are not limited to, the multifunctional E. coli cloning andexpression vectors such as BLUESCRIPT (Stratagene), in which thesequence encoding the polypeptide of interest may be ligated into thevector in frame with sequences for the amino-terminal Met and thesubsequent 7 residues of .beta.-galactosidase so that a hybrid proteinis produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J.Biol. Chem. 264:5503-5509); and the like. pGEX Vectors (Promega,Madison, Wis.) may also be used to express foreign polypeptides asfusion proteins with glutathione S-transferase (GST). In general, suchfusion proteins are soluble and can easily be purified from lysed cellsby adsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems may bedesigned to include heparin, thrombin, or factor XA protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311.Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

An insect system may also be used to express a polypeptide of interest.For example, in one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The sequencesencoding the polypeptide may be cloned into a non-essential region ofthe virus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the polypeptide-encodingsequence will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which the polypeptide of interest may be expressed (Engelhard,E. K. et al. (1994) Proc. Nat'l Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems aregenerally available. For example, in cases where an adenovirus is usedas an expression vector, sequences encoding a polypeptide of interestmay be ligated into an adenovirus transcription/translation complexconsisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus which is capable of expressing thepolypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc.Nat'l Acad. Sci. 81:3655-3659). In addition, transcription enhancers,such as the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf, D. et al.(1994) Results Probl. Cell Differ. 20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation.glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, COS, HeLa, MDCK, HEK293, andW138, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is generally preferred. For example, cell lines which stablyexpress a polynucleotide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1990) Cell 22:817-23) geneswhich can be employed in tk.sup.- or aprt.sup.-cells, respectively.Also, antimetabolite, antibiotic or herbicide resistance can be used asthe basis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Nat'l Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosides,neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14); and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Nat'l Acad. Sci. 85:8047-51). Theuse of visible markers has gained popularity with such markers asanthocyanins, beta-glucuronidase and its substrate GUS, and luciferaseand its substrate luciferin, being widely used not only to identifytransformants, but also to quantify the amount of transient or stableprotein expression attributable to a specific vector system (Rhodes, C.A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding apolypeptide is inserted within a marker gene sequence, recombinant cellscontaining sequences can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with apolypeptide-encoding sequence under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells that contain and express a desiredpolynucleotide sequence may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassayor immunoassay techniques which include, for example, membrane,solution, or chip based technologies for the detection and/orquantification of nucleic acid or protein.

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products, using either polyclonal or monoclonalantibodies specific for the product are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on a given polypeptide may be preferred forsome applications, but a competitive binding assay may also be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul. Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides include oligolabeling,nick translation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the sequences, or any portions thereof may becloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits. Suitable reporter molecules or labels, which may be used includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with a polynucleotide sequence of interest may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides of theinvention may be designed to contain signal sequences which directsecretion of the encoded polypeptide through a prokaryotic or eukaryoticcell membrane. Other recombinant constructions may be used to joinsequences encoding a polypeptide of interest to nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins. Such purification facilitating domains include, butare not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen. San Diego, Calif.) between the purificationdomain and the encoded polypeptide may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing a polypeptide of interest and a nucleic acidencoding 6 histidine residues preceding a thioredoxin or an enterokinasecleavage site. The histidine residues facilitate purification on IMIAC(immobilized metal ion affinity chromatography) as described in Porath,J. et al. (1992, Prot. Exp. Purif. 3:263-281) while the enterokinasecleavage site provides a means for purifying the desired polypeptidefrom the fusion protein. A discussion of vectors which contain fusionproteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol.12:441-453).

In addition to recombinant production methods, polypeptides of theinvention, and fragments thereof, may be produced by direct peptidesynthesis using solid-phase techniques (Merrifield J. (1963) J. Am.Chem. Soc. 85:2149-2154). Protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may be achieved,for example, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Alternatively, various fragments may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule.

Antibody Compositions, Fragments Thereof and Other Binding Agents

According to another aspect, the present invention further providesbinding agents, such as antibodies and antigen-binding fragmentsthereof, that exhibit immunological binding to a tumor polypeptidedisclosed herein, or to a portion, variant or derivative thereof. Anantibody, or antigen-binding fragment thereof, is said to “specificallybind,” “immunogically bind,” and/or is “immunologically reactive” to apolypeptide of the invention if it reacts at a detectable level (within,for example, an ELISA assay) with the polypeptide, and does not reactdetectably with unrelated polypeptides under similar conditions.

Immunological binding, as used in this context, generally refers to thenon-covalent interactions of the type which occur between animmunoglobulin molecule and an antigen for which the immunoglobulin isspecific. The strength, or affinity of immunological bindinginteractions can be expressed in terms of the dissociation constant(K_(d)) of the interaction, wherein a smaller K_(d) represents a greateraffinity. Immunological binding properties of selected polypeptides canbe quantified using methods well known in the art. One such methodentails measuring the rates of antigen-binding site/antigen complexformation and dissociation, wherein those rates depend on theconcentrations of the complex partners, the affinity of the interaction,and on geometric parameters that equally influence the rate in bothdirections. Thus, both the “on rate constant” (K_(on)) and the “off rateconstant” (K_(off)) can be determined by calculation of theconcentrations and the actual rates of association and dissociation. Theratio of K_(off)/K_(on) enables cancellation of all parameters notrelated to affinity, and is thus equal to the dissociation constantK_(d). See, generally, Davies et al. (1990) Annual Rev. Biochem.59:439-473.

An “antigen-binding site,” or “binding portion” of an antibody refers tothe part of the immunoglobulin molecule that participates in antigenbinding. The antigen binding site is formed by amino acid residues ofthe N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”)chains. Three highly divergent stretches within the V regions of theheavy and light chains are referred to as “hypervariable regions” whichare interposed between more conserved flanking stretches known as“framework regions,” or “FRs”. Thus the term “FR” refers to amino acidsequences which are naturally found between and adjacent tohypervariable regions in immunoglobulins. In an antibody molecule, thethree hypervariable regions of a light chain and the three hypervariableregions of a heavy chain are disposed relative to each other in threedimensional space to form an antigen-binding surface. Theantigen-binding surface is complementary to the three-dimensionalsurface of a bound antigen, and the three hypervariable regions of eachof the heavy and light chains are referred to as“complementarity-determining regions,” or “CDRs.”

Binding agents may be further capable of differentiating betweenpatients with and without a cancer, such as prostate cancer, using therepresentative assays provided herein. For example, antibodies or otherbinding agents that bind to a tumor protein will preferably generate asignal indicating the presence of a cancer in at least about 20% ofpatients with the disease, more preferably at least about 30% ofpatients. Alternatively, or in addition, the antibody will generate anegative signal indicating the absence of the disease in at least about90% of individuals without the cancer. To determine whether a bindingagent satisfies this requirement, biological samples (e.g., blood, sera,sputum, urine and/or tumor biopsies) from patients with and without acancer (as determined using standard clinical tests) may be assayed asdescribed herein for the presence of polypeptides that bind to thebinding agent. Preferably, a statistically significant number of sampleswith and without the disease will be assayed. Each binding agent shouldsatisfy the above criteria; however, those of ordinary skill in the artwill recognize that binding agents may be used in combination to improvesensitivity.

Any agent that satisfies the above requirements may be a binding agent.For example, a binding agent may be a ribosome, with or without apeptide component, an RNA molecule or a polypeptide. In a preferredembodiment, a binding agent is an antibody or an antigen-bindingfragment thereof. Antibodies may be prepared by any of a variety oftechniques known to those of ordinary skill in the art. See, e.g.,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988. In general, antibodies can be produced by cell culturetechniques, including the generation of monoclonal antibodies asdescribed herein, or via transfection of antibody genes into suitablebacterial or mammalian cell hosts, in order to allow for the productionof recombinant antibodies. In one technique, an immunogen comprising thepolypeptide is initially injected into any of a wide variety of mammals(e.g., mice, rats, rabbits, sheep or goats). In this step, thepolypeptides of this invention may serve as the immunogen withoutmodification. Alternatively, particularly for relatively shortpolypeptides, a superior immune response may be elicited if thepolypeptide is joined to a carrier protein, such as bovine serum albuminor keyhole limpet hemocyanin. The immunogen is injected into the animalhost, preferably according to a predetermined schedule incorporating oneor more booster immunizations, and the animals are bled periodically.Polyclonal antibodies specific for the polypeptide may then be purifiedfrom such antisera by, for example, affinity chromatography using thepolypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for an antigenic polypeptide of interestmay be prepared, for example, using the technique of Kohler andMilstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.Briefly, these methods involve the preparation of immortal cell linescapable of producing antibodies having the desired specificity (i.e.,reactivity with the polypeptide of interest). Such cell lines may beproduced, for example, from spleen cells obtained from an animalimmunized as described above. The spleen cells are then immortalized by,for example, fusion with a myeloma cell fusion partner, preferably onethat is syngeneic with the immunized animal. A variety of fusiontechniques may be employed. For example, the spleen cells and myelomacells may be combined with a nonionic detergent for a few minutes andthen plated at low density on a selective medium that supports thegrowth of hybrid cells, but not myeloma cells. A preferred selectiontechnique uses HAT (hypoxanthine, aminopterin, thymidine) selection.After a sufficient time, usually about 1 to 2 weeks, colonies of hybridsare observed. Single colonies are selected and their culturesupernatants tested for binding activity against the polypeptide.Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. The polypeptides of this invention may beused in the purification process in, for example, an affinitychromatography step.

A number of therapeutically useful molecules are known in the art whichcomprise antigen-binding sites that are capable of exhibitingimmunological binding properties of an antibody molecule. Theproteolytic enzyme papain preferentially cleaves IgG molecules to yieldseveral fragments, two of which (the “F(ab)” fragments) each comprise acovalent heterodimer that includes an intact antigen-binding site. Theenzyme pepsin is able to cleave IgG molecules to provide severalfragments, including the “F(ab′)₂” fragment which comprises bothantigen-binding sites. An “Fv” fragment can be produced by preferentialproteolytic cleavage of an IgM, and on rare occasions IgG or IgAimmunoglobulin molecule. Fv fragments are, however, more commonlyderived using recombinant techniques known in the art. The Fv fragmentincludes a non-covalent V_(H)::V_(L) heterodimer including anantigen-binding site which retains much of the antigen recognition andbinding capabilities of the native antibody molecule. Inbar et al.(1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976)Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

A single chain Fv (“sFv”) polypeptide is a covalently linkedV_(H)::V_(L) heterodimer which is expressed from a gene fusion includingV_(H)- and V_(L)-encoding genes linked by a peptide-encoding linker.Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. Anumber of methods have been described to discern chemical structures forconverting the naturally aggregated—but chemically separated—light andheavy polypeptide chains from an antibody V region into an sFv moleculewhich will fold into a three dimensional structure substantially similarto the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos.5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778,to Ladner et al.

Each of the above-described molecules includes a heavy chain and a lightchain CDR set, respectively interposed between a heavy chain and a lightchain FR set which provide support to the CDRS and define the spatialrelationship of the CDRs relative to each other. As used herein, theterm “CDR set” refers to the three hypervariable regions of a heavy orlight chain V region. Proceeding from the N-terminus of a heavy or lightchain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3”respectively. An antigen-binding site, therefore, includes six CDRs,comprising the CDR set from each of a heavy and a light chain V region.A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) isreferred to herein as a “molecular recognition unit.” Crystallographicanalysis of a number of antigen-antibody complexes has demonstrated thatthe amino acid residues of CDRs form extensive contact with boundantigen, wherein the most extensive antigen contact is with the heavychain CDR3. Thus, the molecular recognition units are primarilyresponsible for the specificity of an antigen-binding site.

As used herein, the term “FR set” refers to the four flanking amino acidsequences which frame the CDRs of a CDR set of a heavy or light chain Vregion. Some FR residues may contact bound antigen; however, FRs areprimarily responsible for folding the V region into the antigen-bindingsite, particularly the FR residues directly adjacent to the CDRS. WithinFRs, certain amino residues and certain structural features are veryhighly conserved. In this regard, all V region sequences contain aninternal disulfide loop of around 90 amino acid residues. When the Vregions fold into a binding-site, the CDRs are displayed as projectingloop motifs which form an antigen-binding surface. It is generallyrecognized that there are conserved structural regions of FRs whichinfluence the folded shape of the CDR loops into certain “canonical”structures—regardless of the precise CDR amino acid sequence. Further,certain FR residues are known to participate in non-covalent interdomaincontacts which stabilize the interaction of the antibody heavy and lightchains.

A number of “humanized” antibody molecules comprising an antigen-bindingsite derived from a non-human immunoglobulin have been described,including chimeric antibodies having rodent V regions and theirassociated CDRs fused to human constant domains (Winter et al. (1991)Nature 349:293-299; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA86:4220-4224; Shaw et al. (1987) J. Immunol. 138:4534-4538; and Brown etal. (1987) Cancer Res. 47:3577-3583), rodent CDRs grafted into a humansupporting FR prior to fusion with an appropriate human antibodyconstant domain (Riechmann et al. (1988) Nature 332:323-327; Verhoeyenet al. (1988) Science 239:1534-1536; and Jones et al. (1986) Nature321:522-525), and rodent CDRs supported by recombinantly veneered rodentFRs (European Patent Publication No. 519,596, published Dec. 23, 1992).These “humanized” molecules are designed to minimize unwantedimmunological response toward rodent antihuman antibody molecules whichlimits the duration and effectiveness of therapeutic applications ofthose moieties in human recipients.

As used herein, the terms “veneered FRs” and “recombinantly veneeredFRs” refer to the selective replacement of FR residues from, e.g., arodent heavy or light chain V region, with human FR residues in order toprovide a xenogeneic molecule comprising an antigen-binding site whichretains substantially all of the native FR polypeptide foldingstructure. Veneering techniques are based on the understanding that theligand binding characteristics of an antigen-binding site are determinedprimarily by the structure and relative disposition of the heavy andlight chain CDR sets within the antigen-binding surface. Davies et al.(1990) Ann. Rev. Biochem. 59:439-473. Thus, antigen binding specificitycan be preserved in a humanized antibody only wherein the CDRstructures, their interaction with each other, and their interactionwith the rest of the V region domains are carefully maintained. By usingveneering techniques, exterior (e.g., solvent-accessible) FR residueswhich are readily encountered by the immune system are selectivelyreplaced with human residues to provide a hybrid molecule that compriseseither a weakly immunogenic, or substantially non-immunogenic veneeredsurface.

The process of veneering makes use of the available sequence data forhuman antibody variable domains compiled by Kabat et al., in Sequencesof Proteins of Immunological Interest, 4th ed., (U.S. Dept. of Healthand Human Services, U.S. Government Printing Office, 1987), updates tothe Kabat database, and other accessible U.S. and foreign databases(both nucleic acid and protein). Solvent accessibilities of V regionamino acids can be deduced from the known three-dimensional structurefor human and murine antibody fragments. There are two general steps inveneering a murine antigen-binding site. Initially, the FRs of thevariable domains of an antibody molecule of interest are compared withcorresponding FR sequences of human variable domains obtained from theabove-identified sources. The most homologous human V regions are thencompared residue by residue to corresponding murine amino acids. Theresidues in the murine FR which differ from the human counterpart arereplaced by the residues present in the human moiety using recombinanttechniques well known in the art. Residue switching is only carried outwith moieties which are at least partially exposed (solvent accessible),and care is exercised in the replacement of amino acid residues whichmay have a significant effect on the tertiary structure of V regiondomains, such as proline, glycine and charged amino acids.

In this manner, the resultant “veneered” murine antigen-binding sitesare thus designed to retain the murine CDR residues, the residuessubstantially adjacent to the CDRs, the residues identified as buried ormostly buried (solvent inaccessible), the residues believed toparticipate in non-covalent (e.g., electrostatic and hydrophobic)contacts between heavy and light chain domains, and the residues fromconserved structural regions of the FRs which are believed to influencethe “canonical” tertiary structures of the CDR loops. These designcriteria are then used to prepare recombinant nucleotide sequences whichcombine the CDRs of both the heavy and light chain of a murineantigen-binding site into human-appearing FRs that can be used totransfect mammalian cells for the expression of recombinant humanantibodies which exhibit the antigen specificity of the murine antibodymolecule.

In another embodiment of the invention, monoclonal antibodies of thepresent invention may be coupled to one or more therapeutic agents.Suitable agents in this regard include radionuclides, differentiationinducers, drugs, toxins, and derivatives thereof. Preferredradionuclides include ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, and²¹²Bi. Preferred drugs include methotrexate, and pyrimidine and purineanalogs. Preferred differentiation inducers include phorbol esters andbutyric acid. Preferred toxins include ricin, abrin, diptheria toxin,cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, andpokeweed antiviral protein.

A therapeutic agent may be coupled (e.g., covalently bonded) to asuitable monoclonal antibody either directly or indirectly (e.g., via alinker group). A direct reaction between an agent and an antibody ispossible when each possesses a substituent capable of reacting with theother. For example, a nucleophilic group, such as an amino or sulfhydrylgroup, on one may be capable of reacting with a carbonyl-containinggroup, such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide) on the other.

Alternatively, it may be desirable to couple a therapeutic agent and anantibody via a linker group. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on an agent or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of agents, or functional groupson agents, which otherwise would not be possible.

It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g., U.S. Pat. No.4,671,958, to Rodwell et al.

Where a therapeutic agent is more potent when free from the antibodyportion of the immunoconjugates of the present invention, it may bedesirable to use a linker group which is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, toSenter et al.), by hydrolysis of derivatized amino acid side chains(e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serumcomplement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, toRodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789, to Blattler et al.).

It may be desirable to couple more than one agent to an antibody. In oneembodiment, multiple molecules of an agent are coupled to one antibodymolecule. In another embodiment, more than one type of agent may becoupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one agent may be prepared in a varietyof ways. For example, more than one agent may be coupled directly to anantibody molecule, or linkers that provide multiple sites for attachmentcan be used. Alternatively, a carrier can be used.

A carrier may bear the agents in a variety of ways, including covalentbonding either directly or via a linker group. Suitable carriers includeproteins such as albumins (e.g., U.S. Pat. No. 4,507,234, to Kato etal.), peptides and polysaccharides such as aminodextran (e.g., U.S. Pat.No. 4,699,784, to Shih et al.). A carrier may also bear an agent bynoncovalent bonding or by encapsulation, such as within a liposomevesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088). Carriersspecific for radionuclide agents include radiohalogenated smallmolecules and chelating compounds. For example, U.S. Pat. No. 4,735,792discloses representative radiohalogenated small molecules and theirsynthesis. A radionuclide chelate may be formed from chelating compoundsthat include those containing nitrogen and sulfur atoms as the donoratoms for binding the metal, or metal oxide, radionuclide. For example,U.S. Pat. No. 4,673,562, to Davison et al. discloses representativechelating compounds and their synthesis.

T Cell Compositions

The present invention, in another aspect, provides T cells specific fora tumor polypeptide disclosed herein, or for a variant or derivativethereof. Such cells may generally be prepared in vitro or ex vivo, usingstandard procedures. For example, T cells may be isolated from bonemarrow, peripheral blood, or a fraction of bone marrow or peripheralblood of a patient, using a commercially available cell separationsystem, such as the Isolex™ System, available from Nexell Therapeutics,Inc. (Irvine, Calif.; see also U.S. Pat. No. 5,240,856; U.S. Pat. No.5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243). Alternatively, Tcells may be derived from related or unrelated humans, non-humanmammals, cell lines or cultures.

T cells may be stimulated with a polypeptide, polynucleotide encoding apolypeptide and/or an antigen presenting cell (APC) that expresses sucha polypeptide. Such stimulation is performed under conditions and for atime sufficient to permit the generation of T cells that are specificfor the polypeptide of interest. Preferably, a tumor polypeptide orpolynucleotide of the invention is present within a delivery vehicle,such as a microsphere, to facilitate the generation of specific T cells.

T cells are considered to be specific for a polypeptide of the presentinvention if the T cells specifically proliferate, secrete cytokines orkill target cells coated with the polypeptide or expressing a geneencoding the polypeptide. T cell specificity may be evaluated using anyof a variety of standard techniques. For example, within a chromiumrelease assay or proliferation assay, a stimulation index of more thantwo fold increase in lysis and/or proliferation, compared to negativecontrols, indicates T cell specificity. Such assays may be performed,for example, as described in Chen et al., Cancer Res. 54:1065-1070,1994. Alternatively, detection of the proliferation of T cells may beaccomplished by a variety of known techniques. For example, T cellproliferation can be detected by measuring an increased rate of DNAsynthesis (e.g., by pulse-labeling cultures of T cells with tritiatedthymidine and measuring the amount of tritiated thymidine incorporatedinto DNA). Contact with a tumor polypeptide (100 ng/ml-100 μg/ml,preferably 200 ng/ml-25 μg/ml) for 3-7 days will typically result in atleast a two fold increase in proliferation of the T cells. Contact asdescribed above for 2-3 hours should result in activation of the Tcells, as measured using standard cytokine assays in which a two foldincrease in the level of cytokine release (e.g., TNF or IFN-γ) isindicative of T cell activation (see Coligan et al., Current Protocolsin Immunology, vol. 1, Wiley Interscience (Greene 1998)). T cells thathave been activated in response to a tumor polypeptide, polynucleotideor polypeptide-expressing APC may be CD4⁺ and/or CD8⁺. Tumorpolypeptide-specific T cells may be expanded using standard techniques.Within preferred embodiments, the T cells are derived from a patient, arelated donor or an unrelated donor, and are administered to the patientfollowing stimulation and expansion.

For therapeutic purposes, CD4⁺ or CD8⁺ T cells that proliferate inresponse to a tumor polypeptide, polynucleotide or APC can be expandedin number either in vitro or in vivo. Proliferation of such T cells invitro may be accomplished in a variety of ways. For example, the T cellscan be re-exposed to a tumor polypeptide, or a short peptidecorresponding to an immunogenic portion of such a polypeptide, with orwithout the addition of T cell growth factors, such as interleukin-2,and/or stimulator cells that synthesize a tumor polypeptide.Alternatively, one or more T cells that proliferate in the presence ofthe tumor polypeptide can be expanded in number by cloning. Methods forcloning cells are well known in the art, and include limiting dilution.

Pharmaceutical Compositions

In additional embodiments, the present invention concerns formulation ofone or more of the polynucleotide, polypeptide, T-cell and/or antibodycompositions disclosed herein in pharmaceutically-acceptable carriersfor administration to a cell or an animal, either alone, or incombination with one or more other modalities of therapy.

It will be understood that, if desired, a composition as disclosedherein may be administered in combination with other agents as well,such as, e.g., other proteins or polypeptides or variouspharmaceutically-active agents. In fact, there is virtually no limit toother components that may also be included, given that the additionalagents do not cause a significant adverse effect upon contact with thetarget cells or host tissues. The compositions may thus be deliveredalong with various other agents as required in the particular instance.Such compositions may be purified from host cells or other biologicalsources, or alternatively may be chemically synthesized as describedherein. Likewise, such compositions may further comprise substituted orderivatized RNA or DNA compositions.

Therefore, in another aspect of the present invention, pharmaceuticalcompositions are provided comprising one or more of the polynucleotide,polypeptide, antibody, and/or T-cell compositions described herein incombination with a physiologically acceptable carrier. In certainpreferred embodiments, the pharmaceutical compositions of the inventioncomprise immunogenic polynucleotide and/or polypeptide compositions ofthe invention for use in prophylactic and therapeutic vaccineapplications. Vaccine preparation is generally described in, forexample, M. F. Powell and M. J. Newman, eds., “Vaccine Design (thesubunit and adjuvant approach),” Plenum Press (NY, 1995). Generally,such compositions will comprise one or more polynucleotide and/orpolypeptide compositions of the present invention in combination withone or more immunostimulants.

It will be apparent that any of the pharmaceutical compositionsdescribed herein can contain pharmaceutically acceptable salts of thepolynucleotides and polypeptides of the invention. Such salts can beprepared, for example, from pharmaceutically acceptable non-toxic bases,including organic bases (e.g., salts of primary, secondary and tertiaryamines and basic amino acids) and inorganic bases (e.g., sodium,potassium, lithium, ammonium, calcium and magnesium salts).

In another embodiment, illustrative immunogenic compositions, e.g.,vaccine compositions, of the present invention comprise DNA encoding oneor more of the polypeptides as described above, such that thepolypeptide is generated in situ. As noted above, the polynucleotide maybe administered within any of a variety of delivery systems known tothose of ordinary skill in the art. Indeed, numerous gene deliverytechniques are well known in the art, such as those described byRolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, 1998, andreferences cited therein. Appropriate polynucleotide expression systemswill, of course, contain the necessary regulatory DNA regulatorysequences for expression in a patient (such as a suitable promoter andterminating signal). Alternatively, bacterial delivery systems mayinvolve the administration of a bacterium (such asBacillus-Calmette-Guerrin) that expresses an immunogenic portion of thepolypeptide on its cell surface or secretes such an epitope.

Therefore, in certain embodiments, polynucleotides encoding immunogenicpolypeptides described herein are introduced into suitable mammalianhost cells for expression using any of a number of known viral-basedsystems. In one illustrative embodiment, retroviruses provide aconvenient and effective platform for gene delivery systems. A selectednucleotide sequence encoding a polypeptide of the present invention canbe inserted into a vector and packaged in retroviral particles usingtechniques known in the art. The recombinant virus can then be isolatedand delivered to a subject. A number of illustrative retroviral systemshave been described (e.g., U.S. Pat. No. 5,219,740; Miller and Rosman(1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993)Proc. Nat'l Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin(1993) Cur. Opin. Genet. Develop. 3:102-109.

In addition, a number of illustrative adenovirus-based systems have alsobeen described. Unlike retroviruses which integrate into the hostgenome, adenoviruses persist extrachromosomally thus minimizing therisks associated with insertional mutagenesis (Haj-Ahmad and Graham(1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921;Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al.(1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58;Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al. (1993)Human Gene Therapy 4:461-476).

Various adeno-associated virus (AAV) vector systems have also beendeveloped for polynucleotide delivery. AAV vectors can be readilyconstructed using techniques well known in the art. See, e.g., U.S. Pat.Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070and WO 93/03769; Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539;Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shellingand Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp.Med. 179:1867-1875.

Additional viral vectors useful for delivering the polynucleotidesencoding polypeptides of the present invention by gene transfer includethose derived from the pox family of viruses, such as vaccinia virus andavian poxvirus. By way of example, vaccinia virus recombinantsexpressing the novel molecules can be constructed as follows. The DNAencoding a polypeptide is first inserted into an appropriate vector sothat it is adjacent to a vaccinia promoter and flanking vaccinia DNAsequences, such as the sequence encoding thymidine kinase (TK). Thisvector is then used to transfect cells which are simultaneously infectedwith vaccinia. Homologous recombination serves to insert the vacciniapromoter plus the gene encoding the polypeptide of interest into theviral genome. The resulting TK.sup.(−) recombinant can be selected byculturing the cells in the presence of 5-bromodeoxyuridine and pickingviral plaques resistant thereto.

A vaccinia-based infection/transfection system can be conveniently usedto provide for inducible, transient expression or coexpression of one ormore polypeptides described herein in host cells of an organism. In thisparticular system, cells are first infected in vitro with a vacciniavirus recombinant that encodes the bacteriophage T7 RNA polymerase. Thispolymerase displays exquisite specificity in that it only transcribestemplates bearing T7 promoters. Following infection, cells aretransfected with the polynucleotide or polynucleotides of interest,driven by a T7 promoter. The polymerase expressed in the cytoplasm fromthe vaccinia virus recombinant transcribes the transfected DNA into RNAwhich is then translated into polypeptide by the host translationalmachinery. The method provides for high level, transient, cytoplasmicproduction of large quantities of RNA and its translation products. See,e.g., Elroy-Stein and Moss, Proc. Nat'l Acad. Sci. USA (1990)87:6743-6747; Fuerst et al. Proc. Nat'l Acad. Sci. USA (1986)83:8122-8126.

Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses,can also be used to deliver the coding sequences of interest.Recombinant avipox viruses, expressing immunogens from mammalianpathogens, are known to confer protective immunity when administered tonon-avian species. The use of an Avipox vector is particularly desirablein human and other mammalian species since members of the Avipox genuscan only productively replicate in susceptible avian species andtherefore are not infective in mammalian cells. Methods for producingrecombinant Avipoxviruses are known in the art and employ geneticrecombination, as described above with respect to the production ofvaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

Any of a number of alphavirus vectors can also be used for delivery ofpolynucleotide compositions of the present invention, such as thosevectors described in U.S. Pat. Nos. 5,843,723; 6,015,686; 6,008,035 and6,015,694. Certain vectors based on Venezuelan Equine Encephalitis (VEE)can also be used, illustrative examples of which can be found in U.S.Pat. Nos. 5,505,947 and 5,643,576.

Moreover, molecular conjugate vectors, such as the adenovirus chimericvectors described in Michael et al. J. Biol. Chem. (1993) 268:6866-6869and Wagner et al. Proc. Nat'l Acad. Sci. USA (1992) 89:6099-6103, canalso be used for gene delivery under the invention.

Additional illustrative information on these and other known viral-baseddelivery systems can be found, for example, in Fisher-Hoch et al., Proc.Nat'l Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad.Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat.Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No.4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner,Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434,1991; Kolls et al., Proc. Nat'l Acad. Sci. USA 91:215-219, 1994;Kass-Eisler et al., Proc. Nat'l Acad. Sci. USA 90:11498-11502, 1993;Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir.Res. 73:1202-1207, 1993.

In certain embodiments, a polynucleotide may be integrated into thegenome of a target cell. This integration may be in a specific locationand orientation via homologous recombination (gene replacement) or itmay be integrated in a random, non-specific location (geneaugmentation). In yet further embodiments, the polynucleotide may bestably maintained in the cell as a separate, episomal segment of DNA.Such polynucleotide segments or “episomes” encode sequences sufficientto permit maintenance and replication independent of or insynchronization with the host cell cycle. The manner in which theexpression construct is delivered to a cell and where in the cell thepolynucleotide remains is dependent on the type of expression constructemployed.

In another embodiment of the invention, a polynucleotide isadministered/delivered as “naked” DNA, for example as described in Ulmeret al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads, which are efficiently transported intothe cells.

In still another embodiment, a composition of the present invention canbe delivered via a particle bombardment approach, many of which havebeen described. In one illustrative example, gas-driven particleacceleration can be achieved with devices such as those manufactured byPowderject Pharmaceuticals PLC (Oxford, UK) and Powderject Vaccines Inc.(Madison, Wis.), some examples of which are described in U.S. Pat. Nos.5,846,796; 6,010,478; 5,865,796; 5,584,807; and EP Patent No. 0500 799.This approach offers a needle-free delivery approach wherein a drypowder formulation of microscopic particles, such as polynucleotide orpolypeptide particles, are accelerated to high speed within a helium gasjet generated by a hand held device, propelling the particles into atarget tissue of interest.

In a related embodiment, other devices and methods that may be usefulfor gas-driven needle-less injection of compositions of the presentinvention include those provided by Bioject, Inc. (Portland, Oreg.),some examples of which are described in U.S. Pat. Nos. 4,790,824;5,064,413; 5,312,335; 5,383,851; 5,399,163; 5,520,639 and 5,993,412.

According to another embodiment, the pharmaceutical compositionsdescribed herein will comprise one or more immunostimulants in additionto the immunogenic polynucleotide, polypeptide, antibody, T-cell and/orAPC compositions of this invention. An immunostimulant refers toessentially any substance that enhances or potentiates an immuneresponse (antibody and/or cell-mediated) to an exogenous antigen. Onepreferred type of immunostimulant comprises an adjuvant. Many adjuvantscontain a substance designed to protect the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and a stimulatorof immune responses, such as lipid A, Bortadella pertussis orMycobacterium tuberculosis derived proteins. Certain adjuvants arecommercially available as, for example, Freund's Incomplete Adjuvant andComplete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham,Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum)or aluminum phosphate; salts of calcium, iron or zinc; an insolublesuspension of acylated tyrosine; acylated sugars; cationically oranionically derivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such asGM-CSF, interleukin-2, -7, -12, and other like growth factors, may alsobe used as adjuvants.

Within certain embodiments of the invention, the adjuvant composition ispreferably one that induces an immune response predominantly of the Th1type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFβ, IL-2 andIL-12) tend to favor the induction of cell mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction ofhumoral immune responses. Following application of a vaccine as providedherein, a patient will support an immune response that includes Th1- andTh2-type responses. Within a preferred embodiment, in which a responseis predominantly Th1-type, the level of Th1-type cytokines will increaseto a greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann and Coffman, Ann. Rev.Immunol. 7:145-173, 1989.

Certain preferred adjuvants for eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A, together with analuminum salt. MPL® adjuvants are available from Corixa Corporation(Seattle, Wash.; see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611;4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which theCpG dinucleotide is unmethylated) also induce a predominantly Th1response. Such oligonucleotides are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352, 1996. Another preferredadjuvant comprises a saponin, such as Quil A, or derivatives thereof,including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham,Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins.Other preferred formulations include more than one saponin in theadjuvant combinations of the present invention, for example combinationsof at least two of the following group comprising QS21, QS7, Quil A,β-escin, or digitonin.

Alternatively the saponin formulations may be combined with vaccinevehicles composed of chitosan or other polycationic polymers,polylactide and polylactide-co-glycolide particles, poly-N-acetylglucosamine-based polymer matrix, particles composed of polysaccharidesor chemically modified polysaccharides, liposomes and lipid-basedparticles, particles composed of glycerol monoesters, etc. The saponinsmay also be formulated in the presence of cholesterol to formparticulate structures such as liposomes or ISCOMs. Furthermore, thesaponins may be formulated together with a polyoxyethylene ether orester, in either a non-particulate solution or suspension, or in aparticulate structure such as a paucilamelar liposome or ISCOM. Thesaponins may also be formulated with excipients such as Carbopol^(R) toincrease viscosity, or may be formulated in a dry powder form with apowder excipient such as lactose.

In one preferred embodiment, the adjuvant system includes thecombination of a monophosphoryl lipid A and a saponin derivative, suchas the combination of QS21 and 3D-MPL® adjuvant, as described in WO94/00153, or a less reactogenic composition where the QS21 is quenchedwith cholesterol, as described in WO 96/33739. Other preferredformulations comprise an oil-in-water emulsion and tocopherol. Anotherparticularly preferred adjuvant formulation employing QS21, 3D-MPL®adjuvant and tocopherol in an oil-in-water emulsion is described in WO95/17210.

Another enhanced adjuvant system involves the combination of aCpG-containing oligonucleotide and a saponin derivative particularly thecombination of CpG and QS21 is disclosed in WO 00/09159. Preferably theformulation additionally comprises an oil in water emulsion andtocopherol.

Additional illustrative adjuvants for use in the pharmaceuticalcompositions of the invention include Montanide ISA 720 (Seppic,France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59(Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4,available from SmithKline Beecham, Rixensart, Belgium), Detox(Enhanzyn®; Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.)and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as thosedescribed in pending U.S. patent application Ser. Nos. 08/853,826 and09/074,720, the disclosures of which are incorporated herein byreference in their entireties, and polyoxyethylene ether adjuvants suchas those described in WO 99/52549A1.

Other preferred adjuvants include adjuvant molecules of the generalformula

HO(CH₂CH₂O)_(n)-A-R,  (I)

wherein, n is 1-50, A is a bond or —C(O)—, R is C₁₋₅₀ alkyl or PhenylC₁₋₅₀ alkyl.

One embodiment of the present invention consists of a vaccineformulation comprising a polyoxyethylene ether of general formula (I),wherein n is between 1 and 50, preferably 4-24, most preferably 9; the Rcomponent is C₁₋₅₀, preferably C₄-C₂₀ alkyl and most preferably C₁₋₂alkyl, and A is a bond. The concentration of the polyoxyethylene ethersshould be in the range 0.1-20%, preferably from 0.1-10%, and mostpreferably in the range 0.1-1%. Preferred polyoxyethylene ethers areselected from the following group: polyoxyethylene-9-lauryl ether,polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such aspolyoxyethylene lauryl ether are described in the Merck index (12^(th)edition: entry 7717). These adjuvant molecules are described in WO99/52549. The polyoxyethylene ether according to the general formula (I)above may, if desired, be combined with another adjuvant. For example, apreferred adjuvant combination is preferably with CpG as described inthe pending UK patent application GB 9820956.2.

According to another embodiment of this invention, an immunogeniccomposition described herein is delivered to a host via antigenpresenting cells (APCs), such as dendritic cells, macrophages, B cells,monocytes and other cells that may be engineered to be efficient APCs.Such cells may, but need not, be genetically modified to increase thecapacity for presenting the antigen, to improve activation and/ormaintenance of the T cell response, to have anti-tumor effects per seand/or to be immunologically compatible with the receiver (i.e., matchedHLA haplotype). APCs may generally be isolated from any of a variety ofbiological fluids and organs, including tumor and peritumoral tissues,and may be autologous, allogeneic, syngeneic or xenogeneic cells.

Certain preferred embodiments of the present invention use dendriticcells or progenitors thereof as antigen-presenting cells. Dendriticcells are highly potent APCs (Banchereau and Steinman, Nature392:245-251, 1998) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticantitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529,1999). In general, dendritic cells may be identified based on theirtypical shape (stellate in situ, with marked cytoplasmic processes(dendrites) visible in vitro), their ability to take up, process andpresent antigens with high efficiency and their ability to activatenaïve T cell responses. Dendritic cells may, of course, be engineered toexpress specific cell-surface receptors or ligands that are not commonlyfound on dendritic cells in vivo or ex vivo, and such modified dendriticcells are contemplated by the present invention. As an alternative todendritic cells, secreted vesicles antigen-loaded dendritic cells(called exosomes) may be used within a vaccine (see Zitvogel et al.,Nature Med. 4:594-600, 1998).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltratingcells, lymph nodes, spleen, skin, umbilical cord blood or any othersuitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNFβ to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

Dendritic cells are conveniently categorized as “immature” and “mature”cells, which allows a simple way to discriminate between two wellcharacterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,CD86 and 4-1 BB).

APCs may generally be transfected with a polynucleotide of the invention(or portion or other variant thereof) such that the encoded polypeptide,or an immunogenic portion thereof, is expressed on the cell surface.Such transfection may take place ex vivo, and a pharmaceuticalcomposition comprising such transfected cells may then be used fortherapeutic purposes, as described herein. Alternatively, a genedelivery vehicle that targets a dendritic or other antigen presentingcell may be administered to a patient, resulting in transfection thatoccurs in vivo. In vivo and ex vivo transfection of dendritic cells, forexample, may generally be performed using any methods known in the art,such as those described in WO 97/24447, or the gene gun approachdescribed by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.Antigen loading of dendritic cells may be achieved by incubatingdendritic cells or progenitor cells with the tumor polypeptide, DNA(naked or within a plasmid vector) or RNA; or with antigen-expressingrecombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus orlentivirus vectors). Prior to loading, the polypeptide may be covalentlyconjugated to an immunological partner that provides T cell help (e.g.,a carrier molecule). Alternatively, a dendritic cell may be pulsed witha non-conjugated immunological partner, separately or in the presence ofthe polypeptide.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will typically vary depending on the mode ofadministration. Compositions of the present invention may be formulatedfor any appropriate manner of administration, including for example,topical, oral, nasal, mucosal, intravenous, intracranial,intraperitoneal, subcutaneous and intramuscular administration.

Carriers for use within such pharmaceutical compositions arebiocompatible, and may also be biodegradable. In certain embodiments,the formulation preferably provides a relatively constant level ofactive component release. In other embodiments, however, a more rapidrate of release immediately upon administration may be desired. Theformulation of such compositions is well within the level of ordinaryskill in the art using known techniques. Illustrative carriers useful inthis regard include microparticles of poly(lactide-co-glycolide),polyacrylate, latex, starch, cellulose, dextran and the like. Otherillustrative delayed-release carriers include supramolecular biovectors,which comprise a non-liquid hydrophilic core (e.g., a cross-linkedpolysaccharide or oligosaccharide) and, optionally, an external layercomprising an amphiphilic compound, such as a phospholipid (see e.g.,U.S. Pat. No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701and WO 96/06638). The amount of active compound contained within asustained release formulation depends upon the site of implantation, therate and expected duration of release and the nature of the condition tobe treated or prevented.

In another illustrative embodiment, biodegradable microspheres (e.g.,polylactate polyglycolate) are employed as carriers for the compositionsof this invention. Suitable biodegradable microspheres are disclosed,for example, in U.S. Pat. Nos. 4,897,268; 5,075,109; 5,928,647;5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and 5,942,252.Modified hepatitis B core protein carrier systems. such as described inWO/99 40934, and references cited therein, will also be useful for manyapplications. Another illustrative carrier/delivery system employs acarrier comprising particulate-protein complexes, such as thosedescribed in U.S. Pat. No. 5,928,647, which are capable of inducing aclass I-restricted cytotoxic T lymphocyte responses in a host.

The pharmaceutical compositions of the invention will often furthercomprise one or more buffers (e.g., neutral buffered saline or phosphatebuffered saline), carbohydrates (e.g., glucose, mannose, sucrose ordextrans), mannitol, proteins, polypeptides or amino acids such asglycine, antioxidants, bacteriostats, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide), solutes that renderthe formulation isotonic, hypotonic or weakly hypertonic with the bloodof a recipient, suspending agents, thickening agents and/orpreservatives. Alternatively, compositions of the present invention maybe formulated as a lyophilizate.

The pharmaceutical compositions described herein may be presented inunit-dose or multi-dose containers, such as sealed ampoules or vials.Such containers are typically sealed in such a way to preserve thesterility and stability of the formulation until use. In general,formulations may be stored as suspensions, solutions or emulsions inoily or aqueous vehicles. Alternatively, a pharmaceutical compositionmay be stored in a freeze-dried condition requiring only the addition ofa sterile liquid carrier immediately prior to use.

The development of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation, is well known in the art,some of which are briefly discussed below for general purposes ofillustration.

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to an animal. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

The active compounds may even be incorporated with excipients and usedin the form of ingestible tablets, buccal tables, troches, capsules,elixirs, suspensions, syrups, wafers, and the like (see, for example,Mathiowitz et al., Nature 1997 Mar. 27; 386(6623):410-4; Hwang et al.,Crit Rev Ther Drug Carrier Syst 1998; 15(3):243-84; U.S. Pat. No.5,641,515; U.S. Pat. No. 5,580,579 and U.S. Pat. No. 5,792,451).Tablets, troches, pills, capsules and the like may also contain any of avariety of additional components, for example, a binder, such as gumtragacanth, acacia, cornstarch, or gelatin; excipients, such asdicalcium phosphate; a disintegrating agent, such as corn starch, potatostarch, alginic acid and the like; a lubricant, such as magnesiumstearate; and a sweetening agent, such as sucrose, lactose or saccharinmay be added or a flavoring agent, such as peppermint, oil ofwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar, or both.Of course, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compounds may be incorporated intosustained-release preparation and formulations.

Typically, these formulations will contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

For oral administration, the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. Alternatively, the active ingredientmay be incorporated into an oral solution such as one containing sodiumborate, glycerin and potassium bicarbonate, or dispersed in adentifrice, or added in a therapeutically-effective amount to acomposition that may include water, binders, abrasives, flavoringagents, foaming agents, and humectants. Alternatively the compositionsmay be fashioned into a tablet or solution form that may be placed underthe tongue or otherwise dissolved in the mouth.

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally. Suchapproaches are well known to the skilled artisan, some of which arefurther described, for example, in U.S. Pat. No. 5,543,158; U.S. Pat.No. 5,641,515 and U.S. Pat. No. 5,399,363. In certain embodiments,solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations generally will contain a preservative to prevent the growthof microorganisms.

Illustrative pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (for example, see U.S. Pat. No. 5,466,468). In all cases theform must be sterile and must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and/or vegetable oils.Proper fluidity may be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and/or by the use of surfactants. The preventionof the action of microorganisms can be facilitated by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

In one embodiment, for parenteral administration in an aqueous solution,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. Moreover, for human administration, preparationswill of course preferably meet sterility, pyrogenicity, and the generalsafety and purity standards as required by FDA Office of Biologicsstandards.

In another embodiment of the invention, the compositions disclosedherein may be formulated in a neutral or salt form. Illustrativepharmaceutically-acceptable salts include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective.

The carriers can further comprise any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. The phrase “pharmaceutically-acceptable” refersto molecular entities and compositions that do not produce an allergicor similar untoward reaction when administered to a human.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering genes, nucleic acids, and peptidecompositions directly to the lungs via nasal aerosol sprays has beendescribed, e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212.Likewise, the delivery of drugs using intranasal microparticle resins(Takenaga et al., J Controlled Release 1998 Mar. 2; 52(1-2):81-7) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871) are alsowell-known in the pharmaceutical arts. Likewise, illustrativetransmucosal drug delivery in the form of a polytetrafluoroetheylenesupport matrix is described in U.S. Pat. No. 5,780,045.

In certain embodiments, liposomes, nanocapsules, microparticles, lipidparticles, vesicles, and the like, are used for the introduction of thecompositions of the present invention into suitable hostcells/organisms. In particular, the compositions of the presentinvention may be formulated for delivery either encapsulated in a lipidparticle, a liposome, a vesicle, a nanosphere, or a nanoparticle or thelike. Alternatively, compositions of the present invention can be bound,either covalently or non-covalently, to the surface of such carriervehicles.

The formation and use of liposome and liposome-like preparations aspotential drug carriers is generally known to those of skill in the art(see for example, Lasic, Trends Biotechnol 1998 July; 16(7):307-21;Takakura, Nippon Rinsho 1998 March; 56(3):691-5; Chandran et al., IndianJ Exp Biol. 1997 August; 35(8):801-9; Margalit, Crit Rev Ther DrugCarrier Syst. 1995; 12(2-3):233-61; U.S. Pat. No. 5,567,434; U.S. Pat.No. 5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 and U.S.Pat. No. 5,795,587, each specifically incorporated herein by referencein its entirety).

Liposomes have been used successfully with a number of cell types thatare normally difficult to transfect by other procedures, including Tcell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisenet al., J Biol Chem. 1990 Sep. 25; 265(27):16337-42; Muller et al., DNACell Biol. 1990 April; 9(3):221-9). In addition, liposomes are free ofthe DNA length constraints that are typical of viral-based deliverysystems. Liposomes have been used effectively to introduce genes,various drugs, radiotherapeutic agents, enzymes, viruses, transcriptionfactors, allosteric effectors and the like, into a variety of culturedcell lines and animals. Furthermore, the use of liposomes does notappear to be associated with autoimmune responses or unacceptabletoxicity after systemic delivery.

In certain embodiments, liposomes are formed from phospholipids that aredispersed in an aqueous medium and spontaneously form multilamellarconcentric bilayer vesicles (also termed multilamellar vesicles (MLVs).

Alternatively, in other embodiments, the invention provides forpharmaceutically-acceptable nanocapsule formulations of the compositionsof the present invention. Nanocapsules can generally entrap compounds ina stable and reproducible way (see, for example, Quintanar-Guerrero etal., Drug Dev Ind Pharm. 1998 December; 24(12):1113-28). To avoid sideeffects due to intracellular polymeric overloading, such ultrafineparticles (sized around 0.1 μm) may be designed using polymers able tobe degraded in vivo. Such particles can be made as described, forexample, by Couvreur et al., Crit Rev Ther Drug Carrier Syst. 1988;5(1):1-20; zur Muhlen et al., Eur J Pharm Biopharm. 1998 March;45(2):149-55; Zambaux et al. J Controlled Release. 1998 Jan. 2;50(1-3):31-40; and U.S. Pat. No. 5,145,684.

Cancer Therapeutic Methods

In further aspects of the present invention, the pharmaceuticalcompositions described herein may be used for the treatment of cancer,particularly for the immunotherapy of prostate cancer. Within suchmethods, the pharmaceutical compositions described herein areadministered to a patient, typically a warm-blooded animal, preferably ahuman. A patient may or may not be afflicted with cancer. Accordingly,the above pharmaceutical compositions may be used to prevent thedevelopment of a cancer or to treat a patient afflicted with a cancer.Pharmaceutical compositions and vaccines may be administered eitherprior to or following surgical removal of primary tumors and/ortreatment such as administration of radiotherapy or conventionalchemotherapeutic drugs. As discussed above, administration of thepharmaceutical compositions may be by any suitable method, includingadministration by intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal, intradermal, anal, vaginal, topical and oralroutes.

Within certain embodiments, immunotherapy may be active immunotherapy,in which treatment relies on the in vivo stimulation of the endogenoushost immune system to react against tumors with the administration ofimmune response-modifying agents (such as polypeptides andpolynucleotides as provided herein).

Within other embodiments, immunotherapy may be passive immunotherapy, inwhich treatment involves the delivery of agents with establishedtumor-immune reactivity (such as effector cells or antibodies) that candirectly or indirectly mediate antitumor effects and does notnecessarily depend on an intact host immune system. Examples of effectorcells include T cells as discussed above, T lymphocytes (such as CD8⁺cytotoxic T lymphocytes and CD4⁺ T-helper tumor-infiltratinglymphocytes), killer cells (such as Natural Killer cells andlymphokine-activated killer cells), B cells and antigen-presenting cells(such as dendritic cells and macrophages) expressing a polypeptideprovided herein. T cell receptors and antibody receptors specific forthe polypeptides recited herein may be cloned, expressed and transferredinto other vectors or effector cells for adoptive immunotherapy. Thepolypeptides provided herein may also be used to generate antibodies oranti-idiotypic antibodies (as described above and in U.S. Pat. No.4,918,164) for passive immunotherapy.

Effector cells may generally be obtained in sufficient quantities foradoptive immunotherapy by growth in vitro, as described herein. Cultureconditions for expanding single antigen-specific effector cells toseveral billion in number with retention of antigen recognition in vivoare well known in the art. Such in vitro culture conditions typicallyuse intermittent stimulation with antigen, often in the presence ofcytokines (such as IL-2) and non-dividing feeder cells. As noted above,immunoreactive polypeptides as provided herein may be used to rapidlyexpand antigen-specific T cell cultures in order to generate asufficient number of cells for immunotherapy. In particular,antigen-presenting cells, such as dendritic, macrophage, monocyte,fibroblast and/or B cells, may be pulsed with immunoreactivepolypeptides or transfected with one or more polynucleotides usingstandard techniques well known in the art. For example,antigen-presenting cells can be transfected with a polynucleotide havinga promoter appropriate for increasing expression in a recombinant virusor other expression system. Cultured effector cells for use in therapymust be able to grow and distribute widely, and to survive long term invivo. Studies have shown that cultured effector cells can be induced togrow in vivo and to survive long term in substantial numbers by repeatedstimulation with antigen supplemented with IL-2 (see, for example,Cheever et al., Immunological Reviews 157:177, 1997).

Alternatively, a vector expressing a polypeptide recited herein may beintroduced into antigen presenting cells taken from a patient andclonally propagated ex vivo for transplant back into the same patient.Transfected cells may be reintroduced into the patient using any meansknown in the art, preferably in sterile form by intravenous,intracavitary, intraperitoneal or intratumor administration.

Routes and frequency of administration of the therapeutic compositionsdescribed herein, as well as dosage, will vary from individual toindividual, and may be readily established using standard techniques. Ingeneral, the pharmaceutical compositions and vaccines may beadministered by injection (e.g., intracutaneous, intramuscular,intravenous or subcutaneous), intranasally (e.g., by aspiration) ororally. Preferably, between 1 and 10 doses may be administered over a 52week period. Preferably, 6 doses are administered, at intervals of 1month, and booster vaccinations may be given periodically thereafter.Alternate protocols may be appropriate for individual patients. Asuitable dose is an amount of a compound that, when administered asdescribed above, is capable of promoting an anti-tumor immune response,and is at least 10-50% above the basal (i.e., untreated) level. Suchresponse can be monitored by measuring the anti-tumor antibodies in apatient or by vaccine-dependent generation of cytolytic effector cellscapable of killing the patient's tumor cells in vitro. Such vaccinesshould also be capable of causing an immune response that leads to animproved clinical outcome (e.g., more frequent remissions, complete orpartial or longer disease-free survival) in vaccinated patients ascompared to non-vaccinated patients. In general, for pharmaceuticalcompositions and vaccines comprising one or more polypeptides, theamount of each polypeptide present in a dose ranges from about 25 μg to5 mg per kg of host. Suitable dose sizes will vary with the size of thepatient, but will typically range from about 0.1 mL to about 5 mL.

In general, an appropriate dosage and treatment regimen provides theactive compound(s) in an amount sufficient to provide therapeutic and/orprophylactic benefit. Such a response can be monitored by establishingan improved clinical outcome (e.g., more frequent remissions, completeor partial, or longer disease-free survival) in treated patients ascompared to non-treated patients. Increases in preexisting immuneresponses to a tumor protein generally correlate with an improvedclinical outcome. Such immune responses may generally be evaluated usingstandard proliferation, cytotoxicity or cytokine assays, which may beperformed using samples obtained from a patient before and aftertreatment.

Cancer Detection and Diagnostic Compositions, Methods and Kits

In general, a cancer may be detected in a patient based on the presenceof one or more prostate tumor proteins and/or polynucleotides encodingsuch proteins in a biological sample (for example, blood, sera, sputumurine and/or tumor biopsies) obtained from the patient. In other words,such proteins may be used as markers to indicate the presence or absenceof a cancer such as prostate cancer. In addition, such proteins may beuseful for the detection of other cancers. The binding agents providedherein generally permit detection of the level of antigen that binds tothe agent in the biological sample. Polynucleotide primers and probesmay be used to detect the level of mRNA encoding a tumor protein, whichis also indicative of the presence or absence of a cancer. In general, aprostate tumor sequence should be present at a level that is at leastthree fold higher in tumor tissue than in normal tissue

There are a variety of assay formats known to those of ordinary skill inthe art for using a binding agent to detect polypeptide markers in asample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988. In general, the presence or absenceof a cancer in a patient may be determined by (a) contacting abiological sample obtained from a patient with a binding agent; (b)detecting in the sample a level of polypeptide that binds to the bindingagent; and (c) comparing the level of polypeptide with a predeterminedcut-off value.

In a preferred embodiment, the assay involves the use of binding agentimmobilized on a solid support to bind to and remove the polypeptidefrom the remainder of the sample. The bound polypeptide may then bedetected using a detection reagent that contains a reporter group andspecifically binds to the binding agent/polypeptide complex. Suchdetection reagents may comprise, for example, a binding agent thatspecifically binds to the polypeptide or an antibody or other agent thatspecifically binds to the binding agent, such as an anti-immunoglobulin,protein G, protein A or a lectin. Alternatively, a competitive assay maybe utilized, in which a polypeptide is labeled with a reporter group andallowed to bind to the immobilized binding agent after incubation of thebinding agent with the sample. The extent to which components of thesample inhibit the binding of the labeled polypeptide to the bindingagent is indicative of the reactivity of the sample with the immobilizedbinding agent. Suitable polypeptides for use within such assays includefull length prostate tumor proteins and polypeptide portions thereof towhich the binding agent binds, as described above.

The solid support may be any material known to those of ordinary skillin the art to which the tumor protein may be attached. For example, thesolid support may be a test well in a microtiter plate or anitrocellulose or other suitable membrane. Alternatively, the supportmay be a bead or disc, such as glass, fiberglass, latex or a plasticmaterial such as polystyrene or polyvinylchloride. The support may alsobe a magnetic particle or a fiber optic sensor, such as those disclosed,for example, in U.S. Pat. No. 5,359,681. The binding agent may beimmobilized on the solid support using a variety of techniques known tothose of skill in the art, which are amply described in the patent andscientific literature. In the context of the present invention, the term“immobilization” refers to both noncovalent association, such asadsorption, and covalent attachment (which may be a direct linkagebetween the agent and functional groups on the support or may be alinkage by way of a cross-linking agent). Immobilization by adsorptionto a well in a microtiter plate or to a membrane is preferred. In suchcases, adsorption may be achieved by contacting the binding agent, in asuitable buffer, with the solid support for a suitable amount of time.The contact time varies with temperature, but is typically between about1 hour and about 1 day. In general, contacting a well of a plasticmicrotiter plate (such as polystyrene or polyvinylchloride) with anamount of binding agent ranging from about 10 ng to about 10 μg, andpreferably about 100 ng to about 1 μg, is sufficient to immobilize anadequate amount of binding agent.

Covalent attachment of binding agent to a solid support may generally beachieved by first reacting the support with a bifunctional reagent thatwill react with both the support and a functional group, such as ahydroxyl or amino group, on the binding agent. For example, the bindingagent may be covalently attached to supports having an appropriatepolymer coating using benzoquinone or by condensation of an aldehydegroup on the support with an amine and an active hydrogen on the bindingpartner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991,at A12-A13).

In certain embodiments, the assay is a two-antibody sandwich assay. Thisassay may be performed by first contacting an antibody that has beenimmobilized on a solid support, commonly the well of a microtiter plate,with the sample, such that polypeptides within the sample are allowed tobind to the immobilized antibody. Unbound sample is then removed fromthe immobilized polypeptide-antibody complexes and a detection reagent(preferably a second antibody capable of binding to a different site onthe polypeptide) containing a reporter group is added. The amount ofdetection reagent that remains bound to the solid support is thendetermined using a method appropriate for the specific reporter group.

More specifically, once the antibody is immobilized on the support asdescribed above, the remaining protein binding sites on the support aretypically blocked. Any suitable blocking agent known to those ofordinary skill in the art, such as bovine serum albumin or Tween 20™(Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody is thenincubated with the sample, and polypeptide is allowed to bind to theantibody. The sample may be diluted with a suitable diluent, such asphosphate-buffered saline (PBS) prior to incubation. In general, anappropriate contact time (i.e., incubation time) is a period of timethat is sufficient to detect the presence of polypeptide within a sampleobtained from an individual with prostate cancer. Preferably, thecontact time is sufficient to achieve a level of binding that is atleast about 95% of that achieved at equilibrium between bound andunbound polypeptide. Those of ordinary skill in the art will recognizethat the time necessary to achieve equilibrium may be readily determinedby assaying the level of binding that occurs over a period of time. Atroom temperature, an incubation time of about 30 minutes is generallysufficient.

Unbound sample may then be removed by washing the solid support with anappropriate buffer, such as PBS containing 0.1% Tween 20™. The secondantibody, which contains a reporter group, may then be added to thesolid support. Preferred reporter groups include those groups recitedabove.

The detection reagent is then incubated with the immobilizedantibody-polypeptide complex for an amount of time sufficient to detectthe bound polypeptide. An appropriate amount of time may generally bedetermined by assaying the level of binding that occurs over a period oftime. Unbound detection reagent is then removed and bound detectionreagent is detected using the reporter group. The method employed fordetecting the reporter group depends upon the nature of the reportergroup. For radioactive groups, scintillation counting orautoradiographic methods are generally appropriate. Spectroscopicmethods may be used to detect dyes, luminescent groups and fluorescentgroups. Biotin may be detected using avidin, coupled to a differentreporter group (commonly a radioactive or fluorescent group or anenzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.

To determine the presence or absence of a cancer, such as prostatecancer, the signal detected from the reporter group that remains boundto the solid support is generally compared to a signal that correspondsto a predetermined cut-off value. In one preferred embodiment, thecut-off value for the detection of a cancer is the average mean signalobtained when the immobilized antibody is incubated with samples frompatients without the cancer. In general, a sample generating a signalthat is three standard deviations above the predetermined cut-off valueis considered positive for the cancer. In an alternate preferredembodiment, the cut-off value is determined using a Receiver OperatorCurve, according to the method of Sackett et al., Clinical Epidemiology:A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p.106-7. Briefly, in this embodiment, the cut-off value may be determinedfrom a plot of pairs of true positive rates (i.e., sensitivity) andfalse positive rates (100%-specificity) that correspond to each possiblecut-off value for the diagnostic test result. The cut-off value on theplot that is the closest to the upper left-hand corner (i.e., the valuethat encloses the largest area) is the most accurate cut-off value, anda sample generating a signal that is higher than the cut-off valuedetermined by this method may be considered positive. Alternatively, thecut-off value may be shifted to the left along the plot, to minimize thefalse positive rate, or to the right, to minimize the false negativerate. In general, a sample generating a signal that is higher than thecut-off value determined by this method is considered positive for acancer.

In a related embodiment, the assay is performed in a flow-through orstrip test format, wherein the binding agent is immobilized on amembrane, such as nitrocellulose. In the flow-through test, polypeptideswithin the sample bind to the immobilized binding agent as the samplepasses through the membrane. A second, labeled binding agent then bindsto the binding agent-polypeptide complex as a solution containing thesecond binding agent flows through the membrane. The detection of boundsecond binding agent may then be performed as described above. In thestrip test format, one end of the membrane to which binding agent isbound is immersed in a solution containing the sample. The samplemigrates along the membrane through a region containing second bindingagent and to the area of immobilized binding agent. Concentration ofsecond binding agent at the area of immobilized antibody indicates thepresence of a cancer. Typically, the concentration of second bindingagent at that site generates a pattern, such as a line, that can be readvisually. The absence of such a pattern indicates a negative result. Ingeneral, the amount of binding agent immobilized on the membrane isselected to generate a visually discernible pattern when the biologicalsample contains a level of polypeptide that would be sufficient togenerate a positive signal in the two-antibody sandwich assay, in theformat discussed above. Preferred binding agents for use in such assaysare antibodies and antigen-binding fragments thereof. Preferably, theamount of antibody immobilized on the membrane ranges from about 25 ngto about 1 μg, and more preferably from about 50 ng to about 500 ng.Such tests can typically be performed with a very small amount ofbiological sample.

Of course, numerous other assay protocols exist that are suitable foruse with the tumor proteins or binding agents of the present invention.The above descriptions are intended to be exemplary only. For example,it will be apparent to those of ordinary skill in the art that the aboveprotocols may be readily modified to use tumor polypeptides to detectantibodies that bind to such polypeptides in a biological sample. Thedetection of such tumor protein specific antibodies may correlate withthe presence of a cancer.

A cancer may also, or alternatively, be detected based on the presenceof T cells that specifically react with a tumor protein in a biologicalsample. Within certain methods, a biological sample comprising CD4⁺and/or CD8⁺ T cells isolated from a patient is incubated with a tumorpolypeptide, a polynucleotide encoding such a polypeptide and/or an APCthat expresses at least an immunogenic portion of such a polypeptide,and the presence or absence of specific activation of the T cells isdetected. Suitable biological samples include, but are not limited to,isolated T cells. For example, T cells may be isolated from a patient byroutine techniques (such as by Ficoll/Hypaque density gradientcentrifugation of peripheral blood lymphocytes). T cells may beincubated in vitro for 2-9 days (typically 4 days) at 37° C. withpolypeptide (e.g., 5-25 μg/ml). It may be desirable to incubate anotheraliquot of a T cell sample in the absence of tumor polypeptide to serveas a control. For CD4⁺ T cells, activation is preferably detected byevaluating proliferation of the T cells. For CD8⁺ T cells, activation ispreferably detected by evaluating cytolytic activity. A level ofproliferation that is at least two fold greater and/or a level ofcytolytic activity that is at least 20% greater than in disease-freepatients indicates the presence of a cancer in the patient.

As noted above, a cancer may also, or alternatively, be detected basedon the level of mRNA encoding a tumor protein in a biological sample.For example, at least two oligonucleotide primers may be employed in apolymerase chain reaction (PCR) based assay to amplify a portion of atumor cDNA derived from a biological sample, wherein at least one of theoligonucleotide primers is specific for (i.e., hybridizes to) apolynucleotide encoding the tumor protein. The amplified cDNA is thenseparated and detected using techniques well known in the art, such asgel electrophoresis. Similarly, oligonucleotide probes that specificallyhybridize to a polynucleotide encoding a tumor protein may be used in ahybridization assay to detect the presence of polynucleotide encodingthe tumor protein in a biological sample.

To permit hybridization under assay conditions, oligonucleotide primersand probes should comprise an oligonucleotide sequence that has at leastabout 60%, preferably at least about 75% and more preferably at leastabout 90%, identity to a portion of a polynucleotide encoding a tumorprotein of the invention that is at least 10 nucleotides, and preferablyat least 20 nucleotides, in length. Preferably, oligonucleotide primersand/or probes hybridize to a polynucleotide encoding a polypeptidedescribed herein under moderately stringent conditions, as definedabove. Oligonucleotide primers and/or probes which may be usefullyemployed in the diagnostic methods described herein preferably are atleast 10-40 nucleotides in length. In a preferred embodiment, theoligonucleotide primers comprise at least 10 contiguous nucleotides,more preferably at least 15 contiguous nucleotides, of a DNA moleculehaving a sequence as disclosed herein. Techniques for both PCR basedassays and hybridization assays are well known in the art (see, forexample, Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263,1987; Erlich ed., PCR Technology, Stockton Press, NY, 1989).

One preferred assay employs RT-PCR, in which PCR is applied inconjunction with reverse transcription. Typically, RNA is extracted froma biological sample, such as biopsy tissue, and is reverse transcribedto produce cDNA molecules. PCR amplification using at least one specificprimer generates a cDNA molecule, which may be separated and visualizedusing, for example, gel electrophoresis. Amplification may be performedon biological samples taken from a test patient and from an individualwho is not afflicted with a cancer. The amplification reaction may beperformed on several dilutions of cDNA spanning two orders of magnitude.A two-fold or greater increase in expression in several dilutions of thetest patient sample as compared to the same dilutions of thenon-cancerous sample is typically considered positive.

In another embodiment, the compositions described herein may be used asmarkers for the progression of cancer. In this embodiment, assays asdescribed above for the diagnosis of a cancer may be performed overtime, and the change in the level of reactive polypeptide(s) orpolynucleotide(s) evaluated. For example, the assays may be performedevery 24-72 hours for a period of 6 months to 1 year, and thereafterperformed as needed. In general, a cancer is progressing in thosepatients in whom the level of polypeptide or polynucleotide detectedincreases over time. In contrast, the cancer is not progressing when thelevel of reactive polypeptide or polynucleotide either remains constantor decreases with time.

Certain in vivo diagnostic assays may be performed directly on a tumor.One such assay involves contacting tumor cells with a binding agent. Thebound binding agent may then be detected directly or indirectly via areporter group. Such binding agents may also be used in histologicalapplications. Alternatively, polynucleotide probes may be used withinsuch applications.

As noted above, to improve sensitivity, multiple tumor protein markersmay be assayed within a given sample. It will be apparent that bindingagents specific for different proteins provided herein may be combinedwithin a single assay. Further, multiple primers or probes may be usedconcurrently. The selection of tumor protein markers may be based onroutine experiments to determine combinations that results in optimalsensitivity. In addition, or alternatively, assays for tumor proteinsprovided herein may be combined with assays for other known tumorantigens.

In other aspects of the present invention, cell capture technologies maybe used prior to detection to improve the sensitivity of the variousdetection methodologies disclosed herein.

Exemplary cell enrichment methodologies employ immunomagnetic beads thatare coated with specific monoclonal antibodies to surface cell markers,or tetrameric antibody complexes, may be used to first enrich orpositively select cancer cells in a sample. Various commerciallyavailable kits may be used, including Dynabeads® Epithelial Enrich(Dynal Biotech, Oslo, Norway), StemSep™ (StemCell Technologies, Inc.,Vancouver, BC), and RosetteSep (StemCell Technologies). The skilledartisan will recognize that other readily available methodologies andkits may also be suitably employed to enrich or positively selectdesired cell populations.

Dynabeads® Epithelial Enrich contains magnetic beads coated with mAbsspecific for two glycoprotein membrane antigens expressed on normal andneoplastic epithelial tissues. The coated beads may be added to a sampleand the sample then applied to a magnet, thereby capturing the cellsbound to the beads. The unwanted cells are washed away and themagnetically isolated cells eluted from the beads and used in furtheranalyses.

RosetteSep can be used to enrich cells directly from a blood sample andconsists of a cocktail of tetrameric antibodies that target a variety ofunwanted cells and crosslinks them to glycophorin A on red blood cells(RBC) present in the sample, forming rosettes. When centrifuged overFicoll, targeted cells pellet along with the free RBC.

The combination of antibodies in the depletion cocktail determines whichcells will be removed and consequently which cells will be recovered.Antibodies that are available include, but are not limited to: CD2, CD3,CD4, CD5, CD8, CD10, CD11b, CD14, CD15, CD16, CD19, CD20, CD24, CD25,CD29, CD33, CD34, CD36, CD38, CD41, CD45, CD45RA, CD45RO, CD56, CD66B,CD66e, HLA-DR, IgE, and TCRαβ. Additionally, it is contemplated in thepresent invention that mAbs specific for prostate tumor antigens, can bedeveloped and used in a similar manner. For example, mAbs that bind totumor-specific cell surface antigens may be conjugated to magneticbeads, or formulated in a tetrameric antibody complex, and used toenrich or positively select metastatic prostate tumor cells from asample.

Once a sample is enriched or positively selected, cells may be furtheranalyzed. For example, the cells may be lysed and RNA isolated. RNA maythen be subjected to RT-PCR analysis using prostate tumor-specificprimers in a Real-time PCR assay as described herein.

In another aspect of the present invention, cell capture technologiesmay be used in conjunction with real-time PCR to provide a moresensitive tool for detection of metastatic cells expressing prostatetumor antigens. Detection of prostate cancer cells in bone marrowsamples, peripheral blood, biopsies, and other samples is desirable fordiagnosis and prognosis in prostate cancer patients.

The present invention further provides kits for use within any of theabove diagnostic methods. Such kits typically comprise two or morecomponents necessary for performing a diagnostic assay. Components maybe compounds, reagents, containers and/or equipment. For example, onecontainer within a kit may contain a monoclonal antibody or fragmentthereof that specifically binds to a tumor protein. Such antibodies orfragments may be provided attached to a support material, as describedabove. One or more additional containers may enclose elements, such asreagents or buffers, to be used in the assay. Such kits may also, oralternatively, contain a detection reagent as described above thatcontains a reporter group suitable for direct or indirect detection ofantibody binding.

Alternatively, a kit may be designed to detect the level of mRNAencoding a tumor protein in a biological sample. Such kits generallycomprise at least one oligonucleotide probe or primer, as describedabove, that hybridizes to a polynucleotide encoding a tumor protein.Such an oligonucleotide may be used, for example, within a PCR orhybridization assay. Additional components that may be present withinsuch kits include a second oligonucleotide and/or a diagnostic reagentor container to facilitate the detection of a polynucleotide encoding atumor protein.

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Isolation and Characterization of Prostate-SpecificPolypeptides

This Example describes the isolation of certain prostate-specificpolypeptides from a prostate tumor cDNA library.

A human prostate tumor cDNA expression library was constructed fromprostate tumor poly A⁺ RNA using a Superscript Plasmid System for cDNASynthesis and Plasmid Cloning kit (BRL Life Technologies, Gaithersburg,Md. 20897) following the manufacturer's protocol. Specifically, prostatetumor tissues were homogenized with polytron (Kinematica, Switzerland)and total RNA was extracted using Trizol reagent (BRL Life Technologies)as directed by the manufacturer. The poly A⁺ RNA was then purified usinga Qiagen oligotex spin column mRNA purification kit (Qiagen, SantaClarita, Calif. 91355) according to the manufacturer's protocol.First-strand cDNA was synthesized using the NotI/Oligo-dT18 primer.Double-stranded cDNA was synthesized, ligated with EcoRI/BAXI adaptors(Invitrogen, San Diego, Calif.) and digested with NotI. Following sizefractionation with Chroma Spin-1000 columns (Clontech, Palo Alto,Calif.), the cDNA was ligated into the EcoRI/NotI site of pcDNA3.1(Invitrogen) and transformed into ElectroMax E. coli DH10B cells (BRLLife Technologies) by electroporation.

Using the same procedure, a normal human pancreas cDNA expressionlibrary was prepared from a pool of six tissue specimens (Clontech). ThecDNA libraries were characterized by determining the number ofindependent colonies, the percentage of clones that carried insert, theaverage insert size and by sequence analysis. The prostate tumor librarycontained 1.64×10⁷ independent colonies, with 70% of clones having aninsert and the average insert size being 1745 base pairs. The normalpancreas cDNA library contained 3.3×10⁶ independent colonies, with 69%of clones having inserts and the average insert size being 1120 basepairs. For both libraries, sequence analysis showed that the majority ofclones had a full length cDNA sequence and were synthesized from mRNA,with minimal rRNA and mitochondrial DNA contamination.

cDNA library subtraction was performed using the above prostate tumorand normal pancreas cDNA libraries, as described by Hara et al. (Blood,84:189-199, 1994) with some modifications. Specifically, a prostatetumor-specific subtracted cDNA library was generated as follows. Normalpancreas cDNA library (70 μg) was digested with EcoRI, NotI, and SfuI,followed by a filling-in reaction with DNA polymerase Klenow fragment.After phenol-chloroform extraction and ethanol precipitation, the DNAwas dissolved in 100 μl of H₂O, heat-denatured and mixed with 100 μl(100 μg) of Photoprobe biotin (Vector Laboratories, Burlingame, Calif.).As recommended by the manufacturer, the resulting mixture was irradiatedwith a 270 W sunlamp on ice for 20 minutes. Additional Photoprobe biotin(50 μl) was added and the biotinylation reaction was repeated. Afterextraction with butanol five times, the DNA was ethanol-precipitated anddissolved in 23 μl H₂O to form the driver DNA.

To form the tracer DNA, 10 μg prostate tumor cDNA library was digestedwith BamHI and XhoI, phenol chloroform extracted and passed throughChroma spin-400 columns (Clontech). Following ethanol precipitation, thetracer DNA was dissolved in 5 μl H₂O. Tracer DNA was mixed with 15 μldriver DNA and 20 μl of 2× hybridization buffer (1.5 M NaCl/10 mMEDTA/50 mM HEPES pH 7.5/0.2% sodium dodecyl sulfate), overlaid withmineral oil, and heat-denatured completely. The sample was immediatelytransferred into a 68° C. water bath and incubated for 20 hours (longhybridization [LH]). The reaction mixture was then subjected to astreptavidin treatment followed by phenol/chloroform extraction. Thisprocess was repeated three more times. Subtracted DNA was precipitated,dissolved in 12 μl H₂O, mixed with 8 μl driver DNA and 20 μl of 2×hybridization buffer, and subjected to a hybridization at 68° C. for 2hours (short hybridization [SH]). After removal of biotinylateddouble-stranded DNA, subtracted cDNA was ligated into BamHI/XhoI site ofchloramphenicol resistant pBCSK⁺ (Stratagene, La Jolla, Calif. 92037)and transformed into ElectroMax E. coli DH10B cells by electroporationto generate a prostate tumor specific subtracted cDNA library (referredto as “prostate subtraction 1”).

To analyze the subtracted cDNA library, plasmid DNA was prepared from100 independent clones, randomly picked from the subtracted prostatetumor specific library and grouped based on insert size. RepresentativecDNA clones were further characterized by DNA sequencing with a PerkinElmer/Applied Biosystems Division Automated Sequencer Model 373A (FosterCity, Calif.). Six cDNA clones, hereinafter referred to as F1-13, F1-12,F1-16, H1-1, H1-9 and H1-4, were shown to be abundant in the subtractedprostate-specific cDNA library. The determined 3′ and 5′ cDNA sequencesfor F1-12 are provided in SEQ ID NO: 2 and 3, respectively, withdetermined 3′ cDNA sequences for F1-13, F1-16, H1-1, H1-9 and H1-4 beingprovided in SEQ ID NO: 1 and 4-7, respectively.

The cDNA sequences for the isolated clones were compared to knownsequences in the gene bank using the EMBL and GenBank databases (release96). Four of the prostate tumor cDNA clones, F1-13, F1-16, H1-1, andH1-4, were determined to encode the following previously identifiedproteins: prostate specific antigen (PSA), human glandular kallikrein,human tumor expression enhanced gene, and mitochondria cytochrome Coxidase subunit II. H1-9 was found to be identical to a previouslyidentified human autonomously replicating sequence. No significanthomologies to the cDNA sequence for F1-12 were found.

Subsequent studies led to the isolation of a full-length cDNA sequencefor F1-12 (also referred to as P504S). This sequence is provided in SEQID NO: 107, with the corresponding amino acid sequence being provided inSEQ ID NO: 108. cDNA splice variants of P504S are provided in SEQ ID NO:600-605.

To clone less abundant prostate tumor specific genes, cDNA librarysubtraction was performed by subtracting the prostate tumor cDNA librarydescribed above with the normal pancreas cDNA library and with the threemost abundant genes in the previously subtracted prostate tumor specificcDNA library: human glandular kallikrein, prostate specific antigen(PSA), and mitochondria cytochrome C oxidase subunit II. Specifically, 1μg each of human glandular kallikrein, PSA and mitochondria cytochrome Coxidase subunit II cDNAs in pCDNA3.1 were added to the driver DNA andsubtraction was performed as described above to provide a secondsubtracted cDNA library hereinafter referred to as the “subtractedprostate tumor specific cDNA library with spike”.

Twenty-two cDNA clones were isolated from the subtracted prostate tumorspecific cDNA library with spike. The determined 3′ and 5′ cDNAsequences for the clones referred to as J1-17, L1-12, N1-1862, J1-13,J1-19, J1-25, J1-24, K1-58, K1-63, L1-4 and L1-14 are provided in SEQ IDNO: 8-9, 10-11, 12-13, 14-15, 16-17, 18-19, 20-21, 22-23, 24-25, 26-27and 28-29, respectively. The determined 3′ cDNA sequences for the clonesreferred to as J1-12, J1-16, J1-21, K1-48, K1-55, L1-2, L1-6, N1-1858,N1-1860, N1-1861, N1-1864 are provided in SEQ ID NO: 30-40,respectively. Comparison of these sequences with those in the gene bankas described above, revealed no significant homologies to three of thefive most abundant DNA species, (J1-17, L1-12 and N1-1862; SEQ ID NO:8-9, 10-11 and 12-13, respectively). Of the remaining two most abundantspecies, one (J1-12; SEQ ID NO:30) was found to be identical to thepreviously identified human pulmonary surfactant-associated protein, andthe other (K1-48; SEQ ID NO:33) was determined to have some homology toR. norvegicus mRNA for 2-arylpropionyl-CoA epimerase. Of the 17 lessabundant cDNA clones isolated from the subtracted prostate tumorspecific cDNA library with spike, four (J1-16, K1-55, L1-6 and N1-1864;SEQ ID NO:31, 34, 36 and 40, respectively) were found to be identical topreviously identified sequences, two (J1-21 and N1-1860; SEQ ID NO: 32and 38, respectively) were found to show some homology to non-humansequences, and two (L1-2 and N1-1861; SEQ ID NO: 35 and 39,respectively) were found to show some homology to known human sequences.No significant homologies were found to the polypeptides J1-13, J1-19,J1-24, J1-25, K1-58, K1-63, L1-4, L1-14 (SEQ ID NO: 14-15, 16-17, 20-21,18-19, 22-23, 24-25, 26-27, 28-29, respectively).

Subsequent studies led to the isolation of full length cDNA sequencesfor J1-17, L1-12 and N1-1862 (SEQ ID NO: 109-111, respectively). Thecorresponding amino acid sequences are provided in SEQ ID NO: 112-114.L1-12 is also referred to as P501S. A cDNA splice variant of P501S isprovided in SEQ ID NO: 606.

As described below, N1-1862 (also referred to as P503S; SEQ ID NO: 111)was found to show prostate-specific expression. Immunohistochemistry andflow cytometry analysis indicated that P503S is a plasmamembrane-associated molecule. P503S contains four transmembrane domainspredicted to be encoded by amino acids 59-75, 90-106, 107-123 and218-234. P503S contains a cleavable N-terminal signal sequence at aminoacids 1-29. The N-terminus of P503S encoding amino acids 1-58 isinternal, amino acids 76-89 are extracellular, amino acids 124-217 areextracellular and amino acids 235-241 are intracellular. PotentialN-linked glycosylation sites are at amino acids N-141, N154, N-178 andN-184. Glycosylation of P503S has been confirmed experimentally usingenzymes that selectively strip sugar residues from protein and Westernblot analysis. P503S is a member of a family of molecules collectivelyknown as the tetraspanin family or TM4SF (transmembrane 4 superfamily).This family of proteins has been shown to interact, and homo andheterodimerize with, other family members. Furthermore, the TM4SF familymembers associate with a family of adhesion molecules known asintegrins. TM4SF family members have been shown to regulate celladhesion as well as cell proliferation by controlling signaltransduction pathways associated with these physiological responses.

In a further experiment, four additional clones were identified bysubtracting a prostate tumor cDNA library with normal prostate cDNAprepared from a pool of three normal prostate poly A+ RNA (referred toas “prostate subtraction 2”). The determined cDNA sequences for theseclones, hereinafter referred to as U1-3064, U1-3065, V1-3692 and1A-3905, are provided in SEQ ID NO: 69-72, respectively. Comparison ofthe determined sequences with those in the gene bank revealed nosignificant homologies to U1-3065.

A second subtraction with spike (referred to as “prostate subtractionspike 2”) was performed by subtracting a prostate tumor specific cDNAlibrary with spike with normal pancreas cDNA library and further spikedwith PSA, J1-17, pulmonary surfactant-associated protein, mitochondrialDNA, cytochrome c oxidase subunit II, N1-1862, autonomously replicatingsequence, L1-12 and tumor expression enhanced gene. Four additionalclones, hereinafter referred to as V1-3686, R1-2330, 1B-3976 andV1-3679, were isolated. The determined cDNA sequences for these clonesare provided in SEQ ID NO:73-76, respectively. Comparison of thesesequences with those in the gene bank revealed no significant homologiesto V1-3686 and R1-2330.

Further analysis of the three prostate subtractions described above(prostate subtraction 2, subtracted prostate tumor specific cDNA librarywith spike, and prostate subtraction spike 2) resulted in theidentification of sixteen additional clones, referred to as 1G-4736,1G-4738, 1G-4741, 1G-4744, 1G-4734, 1H-4774, 1H-4781, 1H-4785, 1H-4787,1H-4796, 1I-4810, 1I-4811, 1I-4876, 1K-4884 and 1K-4896. The determinedcDNA sequences for these clones are provided in SEQ ID NO: 77-92,respectively. Comparison of these sequences with those in the gene bankas described above, revealed no significant homologies to 1G-4741,1G-4734, 1I-4807, 1I-4876 and 1K-4896 (SEQ ID NO: 79, 81, 87, 90 and 92,respectively). Further analysis of the isolated clones led to thedetermination of extended cDNA sequences for 1G-4736, 1G-4738, 1G-4741,1G-4744, 1H-4774, 1H-4781, 1H-4785, 1H-4787, 1H-4796, 1I-4807, 1I-4876,1K-4884 and 1K-4896, provided in SEQ ID NO: 179-188 and 191-193,respectively, and to the determination of additional partial cDNAsequences for 1I-4810 and 1I-4811, provided in SEQ ID NO: 189 and 190,respectively.

Additional studies with prostate subtraction spike 2 resulted in theisolation of three more clones. Their sequences were determined asdescribed above and compared to the most recent GenBank. All threeclones were found to have homology to known genes, which areCysteine-rich protein, KIAA0242, and KIAA0280 (SEQ ID NO: 317, 319, and320, respectively). Further analysis of these clones by Syntenimicroarray (Synteni, Palo Alto, Calif.) demonstrated that all threeclones were over-expressed in most prostate tumors and prostate BPH, aswell as in the majority of normal prostate tissues tested, but lowexpression in all other normal tissues.

An additional subtraction was performed by subtracting a normal prostatecDNA library with normal pancreas cDNA (referred to as “prostatesubtraction 3”). This led to the identification of six additional clonesreferred to as 1G-4761, 1G-4762, 1H-4766, 1H-4770, 1H-4771 and 1H-4772(SEQ ID NO: 93-98). Comparison of these sequences with those in the genebank revealed no significant homologies to 1G-4761 and 1H-4771 (SEQ IDNO: 93 and 97, respectively). Further analysis of the isolated clonesled to the determination of extended cDNA sequences for 1G-4761,1G-4762, 1H-4766 and 1H-4772 provided in SEQ ID NO: 194-196 and 199,respectively, and to the determination of additional partial cDNAsequences for 1H-4770 and 1H-4771, provided in SEQ ID NO: 197 and 198,respectively.

Subtraction of a prostate tumor cDNA library, prepared from a pool ofpolyA+ RNA from three prostate cancer patients, with a normal pancreascDNA library (prostate subtraction 4) led to the identification of eightclones, referred to as 1D-4297, 1D-4309, 1D.1-4278, 1D-4288, 1D-4283,1D-4304, 1D-4296 and 1D-4280 (SEQ ID NO: 99-107). These sequences werecompared to those in the gene bank as described above. No significanthomologies were found to 1D-4283 and 1D-4304 (SEQ ID NO: 103 and 104,respectively). Further analysis of the isolated clones led to thedetermination of extended cDNA sequences for 1D-4309, 1D.1-4278,1D-4288, 1D-4283, 1D-4304, 1D-4296 and 1D-4280, provided in SEQ ID NO:200-206, respectively.

cDNA clones isolated in prostate subtraction 1 and prostate subtraction2, described above, were colony PCR amplified and their mRNA expressionlevels in prostate tumor, normal prostate and in various other normaltissues were determined using microarray technology (Synteni, Palo Alto,Calif.). Briefly, the PCR amplification products were dotted onto slidesin an array format, with each product occupying a unique location in thearray. mRNA was extracted from the tissue sample to be tested, reversetranscribed, and fluorescent-labeled cDNA probes were generated. Themicroarrays were probed with the labeled cDNA probes, the slides scannedand fluorescence intensity was measured. This intensity correlates withthe hybridization intensity. Two clones (referred to as P509S and P510S)were found to be over-expressed in prostate tumor and normal prostateand expressed at low levels in all other normal tissues tested (liver,pancreas, skin, bone marrow, brain, breast, adrenal gland, bladder,testes, salivary gland, large intestine, kidney, ovary, lung, spinalcord, skeletal muscle and colon). The determined cDNA sequences forP509S and P510S are provided in SEQ ID NO: 223 and 224, respectively.Comparison of these sequences with those in the gene bank as describedabove, revealed some homology to previously identified ESTs.

Additional, studies led to the isolation of the full-length cDNAsequence for P509S. This sequence is provided in SEQ ID NO: 332, withthe corresponding amino acid sequence being provided in SEQ ID NO: 339.Two variant full-length cDNA sequences for P510S are provided in SEQ IDNO: 535 and 536, with the corresponding amino acid sequences beingprovided in SEQ ID NO: 537 and 538, respectively. Additional splicevariants of P510S are provided in SEQ ID NO: 598 and 599.

P510S was found to be expressed in a prostate-specific manner.Immunohistochemistry and flow cytometry analysis indicated that P510S isa plasma membrane-associated molecule. Based on sequence similarity toother multidrug resistance-associated transporter proteins, P510S ispredicted to be a cell surface expressed drug transport protein thatcontains an ATP binding cassette. P510S contains nine transmembranedomains predicted to be encoded by amino acids 135-151, 233-249,330-346, 351-367, 711-727, 775-791, 850-866, 867-883 and 954-970. TheC-terminus of P510S is predicted to be intracellular. Potential N-linkedglycosylation sites are at amino acids N-651, N-690, N-746 and N754,N792, N-1176 and N-1312.

The determined cDNA sequences for additional prostate-specific clonesisolated during characterization of prostate specific cDNA libraries areprovided in SEQ ID NO: 618-689, 691-697 and 709-772. Comparison of thesesequences with those in the public databases revealed no significanthomologies to any of these sequences.

Example 2 Determination of Tissue Specificity of Prostate-SpecificPolypeptides

Using gene specific primers, mRNA expression levels for therepresentative prostate-specific polypeptides F1-16, H1-1, J1-17 (alsoreferred to as P502S), L1-12 (also referred to as P501S), F1-12 (alsoreferred to as P504S) and N1-1862 (also referred to as P503S) wereexamined in a variety of normal and tumor tissues using RT-PCR.

Briefly, total RNA was extracted from a variety of normal and tumortissues using Trizol reagent as described above. First strand synthesiswas carried out using 1-2 μg of total RNA with SuperScript II reversetranscriptase (BRL Life Technologies) at 42° C. for one hour. The cDNAwas then amplified by PCR with gene-specific primers. To ensure thesemi-quantitative nature of the RT-PCR, β-actin was used as an internalcontrol for each of the tissues examined. First, serial dilutions of thefirst strand cDNAs were prepared and RT-PCR assays were performed usingβ-actin specific primers. A dilution was then chosen that enabled thelinear range amplification of the β-actin template and which wassensitive enough to reflect the differences in the initial copy numbers.Using these conditions, the β-actin levels were determined for eachreverse transcription reaction from each tissue. DNA contamination wasminimized by DNase treatment and by assuring a negative PCR result whenusing first strand cDNA that was prepared without adding reversetranscriptase.

mRNA Expression levels were examined in four different types of tumortissue (prostate tumor from 2 patients, breast tumor from 3 patients,colon tumor, lung tumor), and sixteen different normal tissues,including prostate, colon, kidney, liver, lung, ovary, pancreas,skeletal muscle, skin, stomach, testes, bone marrow and brain. F1-16 wasfound to be expressed at high levels in prostate tumor tissue, colontumor and normal prostate, and at lower levels in normal liver, skin andtestes, with expression being undetectable in the other tissuesexamined. H1-1 was found to be expressed at high levels in prostatetumor, lung tumor, breast tumor, normal prostate, normal colon andnormal brain, at much lower levels in normal lung, pancreas, skeletalmuscle, skin, small intestine, bone marrow, and was not detected in theother tissues tested. J1-17 (P502S) and L1-12 (P501S) appear to bespecifically over-expressed in prostate, with both genes being expressedat high levels in prostate tumor and normal prostate but at low toundetectable levels in all the other tissues examined. N1-1862 (P503S)was found to be over-expressed in 60% of prostate tumors and detectablein normal colon and kidney. The RT-PCR results thus indicate that F1-16,H1-1, J1-17 (P502S), N1-1862 (P503S) and L1-12 (P501S) are eitherprostate specific or are expressed at significantly elevated levels inprostate.

Further RT-PCR studies showed that F1-12 (P504S) is over-expressed in60% of prostate tumors, detectable in normal kidney but not detectablein all other tissues tested. Similarly, R1-2330 was shown to beover-expressed in 40% of prostate tumors, detectable in normal kidneyand liver, but not detectable in all other tissues tested. U1-3064 wasfound to be over-expressed in 60% of prostate tumors, and also expressedin breast and colon tumors, but was not detectable in normal tissues.

RT-PCR characterization of R1-2330, U1-3064 and 1D-4279 showed thatthese three antigens are over-expressed in prostate and/or prostatetumors.

Northern analysis with four prostate tumors, two normal prostatesamples, two BPH prostates, and normal colon, kidney, liver, lung,pancrease, skeletal muscle, brain, stomach, testes, small intestine andbone marrow, showed that L1-12 (P501S) is over-expressed in prostatetumors and normal prostate, while being undetectable in other normaltissues tested. J1-17 (P502S) was detected in two prostate tumors andnot in the other tissues tested. N1-1862 (P503S) was found to beover-expressed in three prostate tumors and to be expressed in normalprostate, colon and kidney, but not in other tissues tested. F1-12(P504S) was found to be highly expressed in two prostate tumors and tobe undetectable in all other tissues tested.

The microarray technology described above was used to determine theexpression levels of representative antigens described herein inprostate tumor, breast tumor and the following normal tissues: prostate,liver, pancreas, skin, bone marrow, brain, breast, adrenal gland,bladder, testes, salivary gland, large intestine, kidney, ovary, lung,spinal cord, skeletal muscle and colon. L-1-2 (P501S) was found to beover-expressed in normal prostate and prostate tumor, with someexpression being detected in normal skeletal muscle. Both J1-12 andF1-12 (P504S) were found to be over-expressed in prostate tumor, withexpression being lower or undetectable in all other tissues tested.N1-1862 (P503S) was found to be expressed at high levels in prostatetumor and normal prostate, and at low levels in normal large intestineand normal colon, with expression being undetectable in all othertissues tested. R1-2330 was found to be over-expressed in prostate tumorand normal prostate, and to be expressed at lower levels in all othertissues tested. 1D-4279 was found to be over-expressed in prostate tumorand normal prostate, expressed at lower levels in normal spinal cord,and to be undetectable in all other tissues tested.

Further microarray analysis to specifically address the extent to whichP501S (SEQ ID NO: 110) was expressed in breast tumor revealed moderateover-expression not only in breast tumor, but also in metastatic breasttumor (2/31), with negligible to low expression in normal tissues. Thisdata suggests that P501S may be over-expressed in various breast tumorsas well as in prostate tumors.

The expression levels of 32 ESTs (expressed sequence tags) described byVasmatzis et al. (Proc. Nat'l Acad. Sci. USA 95:300-304, 1998) in avariety of tumor and normal tissues were examined by microarraytechnology as described above. Two of these clones (referred to asP1000C and P1001C) were found to be over-expressed in prostate tumor andnormal prostate, and expressed at low to undetectable levels in allother tissues tested (normal aorta, thymus, resting and activated PBMC,epithelial cells, spinal cord, adrenal gland, fetal tissues, skin,salivary gland, large intestine, bone marrow, liver, lung, dendriticcells, stomach, lymph nodes, brain, heart, small intestine, skeletalmuscle, colon and kidney. The determined cDNA sequences for P1000C andP1001C are provided in SEQ ID NO: 384 and 472, respectively. Thesequence of P1001C was found to show some homology to the previouslyisolated Human mRNA for JM27 protein. Subsequent comparison of thesequence of SEQ ID NO: 384 with sequences in the public databases, ledto the identification of a full-length cDNA sequence of P1000C (SEQ IDNO: 929), which encodes a 492 amino acid sequence. Analysis of the aminoacid sequence using the PSORT II program led to the identification of aputative transmembrane domain from amino acids 84-100. The cDNA sequenceof the open reading frame of P1000C, including the stop codon, isprovided in SEQ ID NO: 930, with the open reading frame without the stopcodon being provided in SEQ ID NO: 931. The full-length amino acidsequence of P1000C is provided in SEQ ID NO: 932. SEQ ID NO: 933 and 934represent amino acids 1-100 and 100-492 of P1000C, respectively.

The expression of the polypeptide encoded by the full length cDNAsequence for F1-12 (also referred to as P504S; SEQ ID NO: 108) wasinvestigated by immunohistochemical analysis. Rabbit-anti-P504Spolyclonal antibodies were generated against the full length P504Sprotein by standard techniques. Subsequent isolation andcharacterization of the polyclonal antibodies were also performed bytechniques well known in the art. Immunohistochemical analysis showedthat the P504S polypeptide was expressed in 100% of prostate carcinomasamples tested (n=5).

The rabbit-anti-P504S polyclonal antibody did not appear to label benignprostate cells with the same cytoplasmic granular staining, but ratherwith light nuclear staining. Analysis of normal tissues revealed thatthe encoded polypeptide was found to be expressed in some, but not allnormal human tissues. Positive cytoplasmic staining withrabbit-anti-P504S polyclonal antibody was found in normal human kidney,liver, brain, colon and lung-associated macrophages, whereas heart andbone marrow were negative.

This data indicates that the P504S polypeptide is present in prostatecancer tissues, and that there are qualitative and quantitativedifferences in the staining between benign prostatic hyperplasia tissuesand prostate cancer tissues, suggesting that this polypeptide may bedetected selectively in prostate tumors and therefore be useful in thediagnosis of prostate cancer.

Example 3 Isolation and Characterization of Prostate-SpecificPolypeptides by PCR-Based Subtraction

A cDNA subtraction library, containing cDNA from normal prostatesubtracted with ten other normal tissue cDNAs (brain, heart, kidney,liver, lung, ovary, placenta, skeletal muscle, spleen and thymus) andthen submitted to a first round of PCR amplification, was purchased fromClontech. This library was subjected to a second round of PCRamplification, following the manufacturer's protocol. The resulting cDNAfragments were subcloned into the vector pT7 Blue T-vector (Novagen,Madison, Wis.) and transformed into XL-1 Blue MRF′ E. coli (Stratagene).DNA was isolated from independent clones and sequenced using a PerkinElmer/Applied Biosystems Division Automated Sequencer Model 373A.

Fifty-nine positive clones were sequenced. Comparison of the DNAsequences of these clones with those in the gene bank, as describedabove, revealed no significant homologies to 25 of these clones,hereinafter referred to as P5, P8, P9, P18, P20, P30, P34, P36, P38,P39, P42, P49, P50, P53, P55, P60, P64, P65, P73, P75, P76, P79 and P84.The determined cDNA sequences for these clones are provided in SEQ IDNO: 41-45, 47-52 and 54-65, respectively. P29, P47, P68, P80 and P82(SEQ ID NO: 46, 53 and 66-68, respectively) were found to show somedegree of homology to previously identified DNA sequences. To the bestof the inventors' knowledge, none of these sequences have beenpreviously shown to be present in prostate.

Further studies employing the sequence of SEQ ID NO: 67 as a probe instandard full-length cloning methods, resulted in the isolation of threecDNA sequences which appear to be splice variants of P80 (also known asP704P). These sequences are provided in SEQ ID NO: 699-701.

Further studies using the PCR-based methodology described above resultedin the isolation of more than 180 additional clones, of which 23 cloneswere found to show no significant homologies to known sequences. Thedetermined cDNA sequences for these clones are provided in SEQ ID NO:115-123, 127, 131, 137, 145, 147-151, 153, 156-158 and 160. Twenty-threeclones (SEQ ID NO: 124-126, 128-130, 132-136, 138-144, 146, 152, 154,155 and 159) were found to show some homology to previously identifiedESTs. An additional ten clones (SEQ ID NO: 161-170) were found to havesome degree of homology to known genes. Larger cDNA clones containingthe P20 sequence represent splice variants of a gene referred to asP703P. The determined DNA sequence for the variants referred to as DE1,DE13 and DE14 are provided in SEQ ID NO: 171, 175 and 177, respectively,with the corresponding amino acid sequences being provided in SEQ ID NO:172, 176 and 178, respectively. The determined cDNA sequence for anextended spliced form of P703 is provided in SEQ ID NO: 225. The DNAsequences for the splice variants referred to as DE2 and DE6 areprovided in SEQ ID NO: 173 and 174, respectively.

mRNA Expression levels for representative clones in tumor tissues(prostate (n=5), breast (n=2), colon and lung) normal tissues (prostate(n=5), colon, kidney, liver, lung (n=2), ovary (n=2), skeletal muscle,skin, stomach, small intestine and brain), and activated andnon-activated PBMC was determined by RT-PCR as described above.Expression was examined in one sample of each tissue type unlessotherwise indicated.

P9 was found to be highly expressed in normal prostate and prostatetumor compared to all normal tissues tested except for normal colonwhich showed comparable expression. P20, a portion of the P703P gene,was found to be highly expressed in normal prostate and prostate tumor,compared to all twelve normal tissues tested. A modest increase inexpression of P20 in breast tumor (n=2), colon tumor and lung tumor wasseen compared to all normal tissues except lung (1 of 2). Increasedexpression of P18 was found in normal prostate, prostate tumor andbreast tumor compared to other normal tissues except lung and stomach. Amodest increase in expression of P5 was observed in normal prostatecompared to most other normal tissues. However, some elevated expressionwas seen in normal lung and PBMC. Elevated expression of P5 was alsoobserved in prostate tumors (2 of 5), breast tumor and one lung tumorsample. For P30, similar expression levels were seen in normal prostateand prostate tumor, compared to six of twelve other normal tissuestested. Increased expression was seen in breast tumors, one lung tumorsample and one colon tumor sample, and also in normal PBMC. P29 wasfound to be over-expressed in prostate tumor (5 of 5) and normalprostate (5 of 5) compared to the majority of normal tissues. However,substantial expression of P29 was observed in normal colon and normallung (2 of 2). P80 was found to be over-expressed in prostate tumor (5of 5) and normal prostate (5 of 5) compared to all other normal tissuestested, with increased expression also being seen in colon tumor.

Further studies resulted in the isolation of twelve additional clones,hereinafter referred to as 10-d8, 10-h10, 11-c8, 7-g6, 8-b5, 8-b6, 8-d4,8-d9, 8-g3, 8-h11, 9-f12 and 9-f3. The determined DNA sequences for10-d8, 10-h10, 11-c8, 8-d4, 8-d9, 8-h11, 9-f12 and 9-f3 are provided inSEQ ID NO: 207, 208, 209, 216, 217, 220, 221 and 222, respectively. Thedetermined forward and reverse DNA sequences for 7-g6, 8-b5, 8-b6 and8-g3 are provided in SEQ ID NO: 210 and 211; 212 and 213; 214 and 215;and 218 and 219, respectively. Comparison of these sequences with thosein the gene bank revealed no significant homologies to the sequence of9-f3. The clones 10-d8, 11-c8 and 8-h11 were found to show some homologyto previously isolated ESTs, while 10-h10, 8-b5, 8-b6, 8-d4, 8-d9, 8-g3and 9-f12 were found to show some homology to previously identifiedgenes. Further characterization of 7-G6 and 8-G3 showed identity to theknown genes PAP and PSA, respectively.

mRNA expression levels for these clones were determined using themicro-array technology described above. The clones 7-G6, 8-G3, 8-B5,8-B6, 8-D4, 8-D9, 9-F3, 9-F12, 9-H3, 10-A2, 10-A4, 11-C9 and 11-F2 werefound to be over-expressed in prostate tumor and normal prostate, withexpression in other tissues tested being low or undetectable. Increasedexpression of 8-F11 was seen in prostate tumor and normal prostate,bladder, skeletal muscle and colon. Increased expression of 10-H10 wasseen in prostate tumor and normal prostate, bladder, lung, colon, brainand large intestine. Increased expression of 9-B1 was seen in prostatetumor, breast tumor, and normal prostate, salivary gland, largeintestine and skin, with increased expression of 11-C8 being seen inprostate tumor, and normal prostate and large intestine.

An additional cDNA fragment derived from the PCR-based normal prostatesubtraction, described above, was found to be prostate specific by bothmicro-array technology and RT-PCR. The determined cDNA sequence of thisclone (referred to as 9-A11) is provided in SEQ ID NO: 226. Comparisonof this sequence with those in the public databases revealed 99%identity to the known gene HOXB13.

Further studies led to the isolation of the clones 8-C6 and 8-H7. Thedetermined cDNA sequences for these clones are provided in SEQ ID NO:227 and 228, respectively. These sequences were found to show somehomology to previously isolated ESTs.

PCR and hybridization-based methodologies were employed to obtain longercDNA sequences for clone P20 (also referred to as P703P), yielding threeadditional cDNA fragments that progressively extend the 5′ end of thegene. These fragments, referred to as P703PDE5, P703P6.26, and P703PX-23(SEQ ID NO: 326, 328 and 330, with the corresponding amino acidsequences being provided in SEQ ID NO: 327, 329 and 331, respectively)contain additional 5′ sequence. P703PDE5 was recovered by screening of acDNA library (#141-26) with a portion of P703P as a probe. P703P6.26 wasrecovered from a mixture of three prostate tumor cDNAs and P703PX_(—)23was recovered from cDNA library (#438-48). Together, the additionalsequences include all of the putative mature serine protease along withpart of the putative signal sequence. The full-length cDNA sequence forP703P is provided in SEQ ID NO: 524, with the corresponding amino acidsequence being provided in SEQ ID NO: 525.

Using computer algorithms, the following regions of P703P were predictedto represent potential HLA A2-binding CTL epitopes: amino acids 164-172of SEQ ID NO: 525 (SEQ ID NO: 866); amino acids 160-168 of SEQ ID NO:525 (SEQ ID NO: 867); amino acids 239-247 of SEQ ID NO: 525 (SEQ ID NO:868); amino acids 118-126 of SEQ ID NO: 525 (SEQ ID NO: 869); aminoacids 112-120 of SEQ ID NO: 525 (SEQ ID NO: 870); amino acids 155-164 ofSEQ ID NO: 525 (SEQ ID NO: 871); amino acids 117-126 of SEQ ID NO: 525(SEQ ID NO: 872); amino acids 164-173 of SEQ ID NO: 525 (SEQ ID NO:873); amino acids 154-163 of SEQ ID NO: 525 (SEQ ID NO: 874); aminoacids 163-172 of SEQ ID NO: 525 (SEQ ID NO: 875); amino acids 58-66 ofSEQ ID NO: 525 (SEQ ID NO: 876); and amino acids 59-67 of SEQ ID NO: 525(SEQ ID NO: 877).

P703P was found to show some homology to previously identifiedproteases, such as thrombin. The thrombin receptor has been shown to bepreferentially expressed in highly metastatic breast carcinoma cells andbreast carcinoma biopsy samples. Introduction of thrombin receptorantisense cDNA has been shown to inhibit the invasion of metastaticbreast carcinoma cells in culture. Antibodies against thrombin receptorinhibit thrombin receptor activation and thrombin-induced plateletactivation. Furthermore, peptides that resemble the receptor's tetheredligand domain inhibit platelet aggregation by thrombin. P703P may play arole in prostate cancer through a protease-activated receptor on thecancer cell or on stromal cells. The potential trypsin-like proteaseactivity of P703P may either activate a protease-activated receptor onthe cancer cell membrane to promote tumorgenesis or activate aprotease-activated receptor on the adjacent cells (such as stromalcells) to secrete growth factors and/or proteases (such as matrixmetalloproteinases) that could promote tumor angiogenesis, invasion andmetastasis. P703P may thus promote tumor progression and/or metastasisthrough the activation of protease-activated receptor. Polypeptides andantibodies that block the P703P-receptor interaction may therefore beusefully employed in the treatment of prostate cancer.

To determine whether P703P expression increases with increased severityof Gleason grade, an indicator of tumor stage, quantitative PCR analysiswas performed on prostate tumor samples with a range of Gleason scoresfrom 5 to >8. The mean level of P703P expression increased withincreasing Gleason score, indicating that P703P expression may correlatewith increased disease severity.

Further studies using a PCR-based subtraction library of a prostatetumor pool subtracted against a pool of normal tissues (referred to asJP: PCR subtraction) resulted in the isolation of thirteen additionalclones, seven of which did not share any significant homology to knownGenBank sequences. The determined cDNA sequences for these seven clones(P711P, P712P, novel 23, P774P, P775P, P710P and P768P) are provided inSEQ ID NO: 307-311, 313 and 315, respectively. The remaining six clones(SEQ ID NO: 316 and 321-325) were shown to share some homology to knowngenes. By microarray analysis, all thirteen clones showed three or morefold over-expression in prostate tissues, including prostate tumors, BPHand normal prostate as compared to normal non-prostate tissues. ClonesP711P, P712P, novel 23 and P768P showed over-expression in most prostatetumors and BPH tissues tested (n=29), and in the majority of normalprostate tissues (n=4), but background to low expression levels in allnormal tissues. Clones P774P, P775P and P710P showed comparatively lowerexpression and expression in fewer prostate tumors and BPH samples, withnegative to low expression in normal prostate.

Further studies led to the isolation of an extended cDNA sequence forP712P (SEQ ID NO: 552). The amino acid sequences encoded by 16 predictedopen reading frames present within the sequence of SEQ ID NO: 552 areprovided in SEQ ID NO: 553-568.

The full-length cDNA for P711P was obtained by employing the partialsequence of SEQ ID NO: 307 to screen a prostate cDNA library.Specifically, a directionally cloned prostate cDNA library was preparedusing standard techniques. One million colonies of this library wereplated onto LB/Amp plates. Nylon membrane filters were used to liftthese colonies, and the cDNAs which were picked up by these filters weredenatured and cross-linked to the filters by UV light. The P711P cDNAfragment of SEQ ID NO: 307 was radio-labeled and used to hybridize withthese filters. Positive clones were selected, and cDNAs were preparedand sequenced using an automatic Perkin Elmer/Applied Biosystemssequencer. The determined full-length sequence of P711P is provided inSEQ ID NO: 382, with the corresponding amino acid sequence beingprovided in SEQ ID NO: 383.

Using PCR and hybridization-based methodologies, additional cDNAsequence information was derived for two clones described above, 11-C9and 9-F3, herein after referred to as P707P and P714P, respectively (SEQID NO: 333 and 334). After comparison with the most recent GenBank,P707P was found to be a splice variant of the known gene HoxB13. Incontrast, no significant homologies to P714P were found. Further studiesemploying the sequence of SEQ ID NO: 334 as a probe in standardfull-length cloning methods, resulted in an extended cDNA sequence forP714P. This sequence is provided in SEQ ID NO: 698. This sequence wasfound to show some homology to the gene that encodes human ribosomalL23A protein.

Clones 8-B3, P89, P98, P130 and P201 (as disclosed in U.S. patentapplication Ser. No. 09/020,956, filed Feb. 9, 1998) were found to becontained within one contiguous sequence, referred to as P705P (cDNAsequence provided in SEQ ID NO: 335, with the amino acid sequence beingprovided in SEQ ID NO: 336), which was determined to be a splice variantof the known gene NKX 3.1. Subsequent studies led to the isolation of acorrected cDNA sequence for P705P. The coding region of this sequence isprovided in SEQ ID NO: 948.

Further studies on P775P resulted in the isolation of four additionalsequences (SEQ ID NO: 473-476) which are all splice variants of theP775P gene. The sequence of SEQ ID NO: 474 was found to contain two openreading frames (ORFs). The amino acid sequences encoded by these ORFsare provided in SEQ ID NO: 477 and 478. The cDNA sequence of SEQ ID NO:475 was found to contain an ORF which encodes the amino acid sequence ofSEQ ID NO: 479. The cDNA sequence of SEQ ID NO: 473 was found to containfour ORFs. The amino acid sequences encoded by these ORFs are providedin SEQ ID NO: 480-483. Additional splice variants of P775P are providedin SEQ ID NO: 593-597.

Subsequent studies led to the identification of a genomic region onchromosome 22q11.2, known as the Cat Eye Syndrome region, that containsthe five prostate genes P704P, P712P, P774P, P775P and B305D. Therelative location of each of these five genes within the genomic regionis shown in FIG. 10. This region may therefore be associated withmalignant tumors, and other potential tumor genes may be containedwithin this region. These studies also led to the identification of apotential open reading frame (ORF) for P775P (provided in SEQ ID NO:533), which encodes the amino acid sequence of SEQ ID NO: 534.

Comparison of the clone of SEQ ID NO: 325 (referred to as P558S) withsequences in the GenBank and GeneSeq DNA databases showed that P558S isidentical to the prostate-specific transglutaminase gene, which is knownto have two forms. The full-length sequences for the two forms areprovided in SEQ ID NO: 773 and 774, with the corresponding amino acidsequences being provided in SEQ ID NO: 775 and 776, respectively. ThecDNA sequence of SEQ ID NO: 774 has a 15 pair base insert, resulting ina 5 amino acid insert in the corresponding amino acid sequence (SEQ IDNO: 776). This insert is not present in the sequence of SEQ ID NO: 773.

Further studies on P768P (SEQ ID NO: 315) led to the identification ofthe putative full-length open reading frame (ORF). The cDNA sequence ofthe ORF with stop codon is provided in SEQ ID NO: 907. The cDNA sequenceof the ORF without stop codon is provided in SEQ ID NO: 908, with thecorresponding amino acid sequence being provided in SEQ ID NO: 909. Thissequence was found to show 86% identity to a rat calcium transporterprotein, indicating that P768P may represent a human calcium transporterprotein. The locations of transmembrane domains within P768P werepredicted using the PSORT II computer algorithm. Six transmembranedomains were predicted at amino acid positions 118-134, 172-188,211-227, 230-246, 282-298 and 348-364. The amino acid sequences of SEQID NO: 910-915 represent amino acids 1-134, 135-188, 189-227, 228-246,247-298 and 299-511 of P768P, respectively.

Example 4 Synthesis of Polypeptides

Polypeptides may be synthesized on a Perkin Elmer/Applied Biosystems430A peptide synthesizer using FMOC chemistry with HPTU(O-Benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate)activation. A Gly-Cys-Gly sequence may be attached to the amino terminusof the peptide to provide a method of conjugation, binding to animmobilized surface, or labeling of the peptide. Cleavage of thepeptides from the solid support may be carried out using the followingcleavage mixture: trifluoroaceticacid:ethanedithiol:thioanisole:water:phenol (40:1:2:2:3). After cleavingfor 2 hours, the peptides may be precipitated in coldmethyl-t-butyl-ether. The peptide pellets may then be dissolved in watercontaining 0.1% trifluoroacetic acid (TFA) and lyophilized prior topurification by C18 reverse phase HPLC. A gradient of 0%-60%acetonitrile (containing 0.1% TFA) in water (containing 0.1% TFA) may beused to elute the peptides. Following lyophilization of the purefractions, the peptides may be characterized using electrospray or othertypes of mass spectrometry and by amino acid analysis.

Example 5 Further Isolation and Characterization of Prostate-SpecificPolypeptides by PCR-Based Subtraction

A cDNA library generated from prostate primary tumor mRNA as describedabove was subtracted with cDNA from normal prostate. The subtraction wasperformed using a PCR-based protocol (Clontech), which was modified togenerate larger fragments. Within this protocol, tester and driverdouble stranded cDNA were separately digested with five restrictionenzymes that recognize six-nucleotide restriction sites (MluI, MscI,PvuII, SalI and StuI). This digestion resulted in an average cDNA sizeof 600 bp, rather than the average size of 300 bp that results fromdigestion with RsaI according to the Clontech protocol. Thismodification did not affect the subtraction efficiency. Two testerpopulations were then created with different adapters, and the driverlibrary remained without adapters.

The tester and driver libraries were then hybridized using excess drivercDNA. In the first hybridization step, driver was separately hybridizedwith each of the two tester cDNA populations. This resulted inpopulations of (a) unhybridized tester cDNAs, (b) tester cDNAshybridized to other tester cDNAs, (c) tester cDNAs hybridized to drivercDNAs and (d) unhybridized driver cDNAs. The two separate hybridizationreactions were then combined, and rehybridized in the presence ofadditional denatured driver cDNA. Following this second hybridization,in addition to populations (a) through (d), a fifth population (e) wasgenerated in which tester cDNA with one adapter hybridized to testercDNA with the second adapter. Accordingly, the second hybridization stepresulted in enrichment of differentially expressed sequences which couldbe used as templates for PCR amplification with adaptor-specificprimers.

The ends were then filled in, and PCR amplification was performed usingadaptor-specific primers. Only population (e), which contained testercDNA that did not hybridize to driver cDNA, was amplified exponentially.A second PCR amplification step was then performed, to reduce backgroundand further enrich differentially expressed sequences.

This PCR-based subtraction technique normalizes differentially expressedcDNAs so that rare transcripts that are overexpressed in prostate tumortissue may be recoverable. Such transcripts would be difficult torecover by traditional subtraction methods.

In addition to genes known to be overexpressed in prostate tumor,seventy-seven further clones were identified. Sequences of these partialcDNAs are provided in SEQ ID NO: 29 to 305. Most of these clones had nosignificant homology to database sequences. Exceptions were JPTPN23 (SEQID NO: 231; similarity to pig valosin-containing protein), JPTPN30 (SEQID NO: 234; similarity to rat mRNA for proteasome subunit), JPTPN45 (SEQID NO: 243; similarity to rat norvegicus cytosolic NADP-dependentisocitrate dehydrogenase), JPTPN46 (SEQ ID NO: 244; similarity to humansubclone H8 4 d4 DNA sequence), JP1D6 (SEQ ID NO: 265; similarity to G.gallus dynein light chain-A), JP8D6 (SEQ ID NO: 288; similarity to humanBAC clone RG016J04), JP8F5 (SEQ ID NO: 289; similarity to human subcloneH8 3 b5 DNA sequence), and JP8E9 (SEQ ID NO: 299; similarity to humanAlu sequence).

Additional studies using the PCR-based subtraction library consisting ofa prostate tumor pool subtracted against a normal prostate pool(referred to as PT-PN PCR subtraction) yielded three additional clones.Comparison of the cDNA sequences of these clones with the most recentrelease of GenBank revealed no significant homologies to the two clonesreferred to as P715P and P767P (SEQ ID NO: 312 and 314). The remainingclone was found to show some homology to the known gene KIAA0056 (SEQ IDNO: 318). Using microarray analysis to measure mRNA expression levels invarious tissues, all three clones were found to be over-expressed inprostate tumors and BPH tissues. Specifically, clone P715P wasover-expressed in most prostate tumors and BPH tissues by a factor ofthree or greater, with elevated expression seen in the majority ofnormal prostate samples and in fetal tissue, but negative to lowexpression in all other normal tissues. Clone P767P was over-expressedin several prostate tumors and BPH tissues, with moderate expressionlevels in half of the normal prostate samples, and background to lowexpression in all other normal tissues tested.

Further analysis, by microarray as described above, of the PT-PN PCRsubtraction library and of a DNA subtraction library containing cDNAfrom prostate tumor subtracted with a pool of normal tissue cDNAs, ledto the isolation of 27 additional clones (SEQ ID NO: 340-365 and 381)which were determined to be over-expressed in prostate tumor. The clonesof SEQ ID NO: 341, 342, 345, 347, 348, 349, 351, 355-359, 361, 362 and364 were also found to be expressed in normal prostate. Expression ofall 26 clones in a variety of normal tissues was found to be low orundetectable, with the exception of P544S (SEQ ID NO: 356) which wasfound to be expressed in small intestine. Of the 26 clones, 11 (SEQ IDNO: 340-349 and 362) were found to show some homology to previouslyidentified sequences. No significant homologies were found to the clonesof SEQ ID NO: 350, 351, 353-361, and 363-365.

Comparison of the sequence of SEQ ID NO: 362 with sequences in theGenBank and GeneSeq DNA databases showed that this clone (referred to asP788P) is identical to GeneSeq Accession No. X27262, which encodes aprotein found in the GeneSeq protein Accession No. Y00931. The fulllength cDNA sequence of P788P is shown in SEQ ID NO: 777, with thecorresponding amino acid being shown in SEQ ID NO: 778. Subsequently, afull-length cDNA sequence for P788P that contains polymorphisms notfound in the sequence of SEQ ID NO: 779, was cloned multiple times byPCR amplification from cDNA prepared from several RNA templates fromthree individuals. This determined cDNA sequence of this polymorphicvariant of P788P is provided in SEQ ID NO: 779, with the correspondingamino acid sequence being provided in SEQ ID NO: 780. The sequence ofSEQ ID NO: 780 differs from that of SEQ ID NO: 778 by six amino acidresidues. The P788P protein has 7 potential transmembrane domains at theC-terminal portion and is predicted to be a plasma membrane protein withan extracellular N-terminal region.

Further studies on the clone of SEQ ID NO: 352 (referred to as P790P)led to the isolation of the full-length cDNA sequence of SEQ ID NO: 526.The corresponding amino acid is provided in SEQ ID NO: 527. Data fromtwo quantitative PCR experiments indicated that P790P is over-expressedin 11/15 tested prostate tumor samples and is expressed at low levels inspinal cord, with no expression being seen in all other normal samplestested. Data from further PCR experiments and microarray experimentsshowed over-expression in normal prostate and prostate tumor with littleor no expression in other tissues tested. P790P was subsequently foundto show significant homology to a previously identified G-proteincoupled prostate tissue receptor.

Additional studies on the clone of SEQ ID NO: 354 (referred to as P776P)led to the isolation of an extended cDNA sequence, provided in SEQ IDNO: 569. The determined cDNA sequences of three additional splicevariants of P776P are provided in SEQ ID NO: 570-572. The amino acidsequences encoded by two predicted open reading frames (ORFs) containedwithin SEQ ID NO: 570, one predicted ORF contained within SEQ ID NO:571, and 11 predicted ORFs contained within SEQ ID NO: 569, are providedin SEQ ID NO: 573-586, respectively. Further studies led to theisolation of the full-length sequence for the clone of SEQ ID NO: 570(provided in SEQ ID NO: 880). Full-length cloning efforts on the cloneof SEQ ID NO: 571 led to the isolation of two sequences (provided in SEQID NO: 881 and 882), representing a single clone, that are identicalwith the exception of a polymorphic insertion/deletion at position 1293.Specifically, the clone of SEQ ID NO: 882 (referred to as clone F1) hasa C at position 1293. The clone of SEQ ID NO: 881 (referred to as cloneF2) has a single base pair deletion at position 1293. The predictedamino acid sequences encoded by 5 open reading frames located within SEQID NO: 880 are provided in SEQ ID NO: 883-887, with the predicted aminoacid sequences encoded by the clone of SEQ ID NO: 881 and 882 beingprovided in SEQ ID NO: 888-893.

Comparison of the cDNA sequences for the clones P767P (SEQ ID NO: 314)and P777P (SEQ ID NO: 350) with sequences in the GenBank human ESTdatabase showed that the two clones matched many EST sequences incommon, suggesting that P767P and P777P may represent the same gene. ADNA consensus sequence derived from a DNA sequence alignment of P767P,P777P and multiple EST clones is provided in SEQ ID NO: 587. The aminoacid sequences encoded by three putative ORFs located within SEQ ID NO:587 are provided in SEQ ID NO: 588-590.

The clone of SEQ ID NO: 342 (referred to as P789P) was found to showhomology to a previously identified gene. The full length cDNA sequencefor P789P and the corresponding amino acid sequence are provided in SEQID NO: 878 and 879, respectively.

Comparison of the sequence of SEQ ID NO: 355 (referred to as P780P) withsequences in the Geneseq DNA database, revealed that P780P shares 100%sequence identity with GeneSEQ accessions Z29049 and Z29050 (SEQ ID NO:944 and 945, respectively). Z29049 and Z29050 differ from each other inthat Z29049 contains a 714 bp deletion relative to Z29050. Thus, Z29049and Z29050 appear to represent 2 alternative splice forms of the geneP780P. Z29049 encodes a protein that is 379 amino acids in length(GeneSeq Accession number Y15155; SEQ ID NO: 946). Z29050 encodes aprotein that is 617 amino acids in length (GeneSeq Accession numberY15156; SEQ ID NO: 947). These two proteins are identical with theexception that Y15155 contains a 238 amino acid deletion relative toY15156. This amino acid deletion corresponds to the 714 bp deletion inZ29049.

Example 6 Peptide Priming of Mice and Propagation of CTL Lines

6.1. This Example illustrates the preparation of a cytotoxic Tlymphocyte (CTL) cell line specific for cells expressing the P502S gene.

Mice expressing the transgene for human HLA A2Kb (provided by Dr L.Sherman, The Scripps Research Institute, La Jolla, Calif.) wereimmunized with P2S#12 peptide (VLGWVAEL; SEQ ID NO: 306), which isderived from the P502S gene (also referred to herein as J1-17, SEQ IDNO: 8), as described by Theobald et al., Proc. Nat'l Acad. Sci. USA92:11993-11997, 1995 with the following modifications. Mice wereimmunized with 100 μg of P2S#12 and 120 μg of an I-A^(b) binding peptidederived from hepatitis B Virus protein emulsified in incomplete Freund'sadjuvant. Three weeks later these mice were sacrificed and single cellsuspensions prepared. Cells were then resuspended at 6×10⁶ cells/ml incomplete media (RPMI-1640; Gibco BRL, Gaithersburg, Md.) containing 10%FCS, 2 mM Glutamine (Gibco BRL), sodium pyruvate (Gibco BRL),non-essential amino acids (Gibco BRL), 2×10⁻⁵ M 2-mercaptoethanol, 50U/ml penicillin and streptomycin, and cultured in the presence ofirradiated (3000 rads) P2S#12-pulsed (5 mg/ml P2S#12 and 10 mg/mlβ2-microglobulin) LPS blasts (A2 transgenic spleens cells cultured inthe presence of 7 μg/ml dextran sulfate and 25 μg/ml LPS for 3 days).Six days later, cells (5×10⁵/ml) were restimulated with 2.5×10⁶/mlpeptide pulsed irradiated (20,000 rads) EL4A2Kb cells (Sherman et al,Science 258:815-818, 1992) and 3×10⁶/ml A2 transgenic spleen feedercells. Cells were cultured in the presence of 20 U/ml IL-2. Cellscontinued to be restimulated on a weekly basis as described, inpreparation for cloning the line.

P2S#12 line was cloned by limiting dilution analysis with peptide pulsedEL4 A2Kb tumor cells (1×10⁴ cells/well) as stimulators and A2 transgenicspleen cells as feeders (5×10⁵ cells/well) grown in the presence of 30U/ml IL-2. On day 14, cells were restimulated as before. On day 21,clones that were growing were isolated and maintained in culture.Several of these clones demonstrated significantly higher reactivity(lysis) against human fibroblasts (HLA A2Kb expressing) transduced withP502S than against control fibroblasts. An example is presented in FIG.1.

This data indicates that P2S #12 represents a naturally processedepitope of the P502S protein that is expressed in the context of thehuman HLA A2Kb molecule.

6.2. This Example illustrates the preparation of murine CTL lines andCTL clones specific for cells expressing the P501S gene.

This series of experiments were performed similarly to that describedabove. Mice were immunized with the P1S#10 peptide (SEQ ID NO: 337),which is derived from the P501S gene (also referred to herein as L1-12,SEQ ID NO: 110). The P1S#10 peptide was derived by analysis of thepredicted polypeptide sequence for P501S for potential HLA-A2 bindingsequences as defined by published HLA-A2 binding motifs (Parker, K C, etal, J. Immunol., 152:163, 1994). P1S#10 peptide was synthesized asdescribed in Example 4, and empirically tested for HLA-A2 binding usinga T cell based competition assay. Predicted A2 binding peptides weretested for their ability to compete HLA-A2 specific peptide presentationto an HLA-A2 restricted CTL clone (D150M58), which is specific for theHLA-A2 binding influenza matrix peptide fluM58. D150M58 CTL secretes TNFin response to self-presentation of peptide fluM58. In the competitionassay, test peptides at 100-200 μg/ml were added to cultures of D150M58CTL in order to bind HLA-A2 on the CTL. After thirty minutes, CTLcultured with test peptides, or control peptides, were tested for theirantigen dose response to the fluM58 peptide in a standard TNF bioassay.As shown in FIG. 3, peptide P1S#10 competes with HLA-A2 restrictedpresentation of fluM58, demonstrating that peptide P1S#10 binds HLA-A2.

Mice expressing the transgene for human HLA A2Kb were immunized asdescribed by Theobald et al. (Proc. Nat'l Acad. Sci. USA 92:11993-11997,1995) with the following modifications. Mice were immunized with 62.5 μgof P1S #10 and 120 μg of an I-A^(b) binding peptide derived fromHepatitis B Virus protein emulsified in incomplete Freund's adjuvant.Three weeks later these mice were sacrificed and single cell suspensionsprepared using a nylon mesh. Cells were then resuspended at 6×10⁶cells/ml in complete media (as described above) and cultured in thepresence of irradiated (3000 rads) P1S#10-pulsed (2 μg/ml P1S#10 and 10mg/ml β2-microglobulin) LPS blasts (A2 transgenic spleens cells culturedin the presence of 7 μg/ml dextran sulfate and 25 μg/ml LPS for 3 days).Six days later cells (5×10⁵/ml) were restimulated with 2.5×10⁶/mlpeptide-pulsed irradiated (20,000 rads) EL4A2Kb cells, as describedabove, and 3×10⁶/ml A2 transgenic spleen feeder cells. Cells werecultured in the presence of 20 U/ml IL-2. Cells were restimulated on aweekly basis in preparation for cloning. After three rounds of in vitrostimulations, one line was generated that recognized P1S#10-pulsedJurkat A2Kb targets and P501S-transduced Jurkat targets as shown in FIG.4.

A P1S#1-specific CTL line was cloned by limiting dilution analysis withpeptide pulsed EL4 A2Kb tumor cells (1×10⁴ cells/well) as stimulatorsand A2 transgenic spleen cells as feeders (5×10⁵ cells/well) grown inthe presence of 30 U/ml IL-2. On day 14, cells were restimulated asbefore. On day 21, viable clones were isolated and maintained inculture. As shown in FIG. 5, five of these clones demonstrated specificcytolytic reactivity against P501S-transduced Jurkat A2Kb targets. Thisdata indicates that P1S#10 represents a naturally processed epitope ofthe P501S protein that is expressed in the context of the human HLA-A2.1molecule.

Example 7 Priming of CTL In Vivo Using Naked DNA Immunization with aProstate Antigen

The prostate-specific antigen L1-12, as described above, is alsoreferred to as P501S. HLA A2Kb Tg mice (provided by Dr L. Sherman, TheScripps Research Institute, La Jolla, Calif.) were immunized with 100 μgP501S in the vector VR1012 either intramuscularly or intradermally. Themice were immunized three times, with a two week interval betweenimmunizations. Two weeks after the last immunization, immune spleencells were cultured with Jurkat A2Kb-P501S transduced stimulator cells.CTL lines were stimulated weekly. After two weeks of in vitrostimulation, CTL activity was assessed against P501S transduced targets.Two out of 8 mice developed strong anti-P501S CTL responses. Theseresults demonstrate that P501S contains at least one naturally processedHLA-A2-restricted CTL epitope.

In subsequent experiments, mice were immunized with either P501S in thevector VR1012 as described above or with a P501S-adenovirus construct at1×10⁸ pfu/animal. Three weeks later, animals received a boostimmunization, with animals that were primed with P501S alone beingboosted with either P501S alone or P501S-adenovirus. Spleen cells wereharvested at 3 and 6 weeks post-immunization and cultured withirradiated Jurkat A2Kb cells (day 0). CTL activity was assayed on day 6,spleen cells restimulated on day 7, and a secondary CTL assay wasperformed on day 13. IgG1 and IgG2a responses against the N-terminalregion of P501S (amino acids 36-325 of SEQ ID NO: 113) and against atruncated form of P501S (amino acids 342-538 of SEQ ID NO: 113) wereassayed using ELISA. The P501S-adenovirus construct effectively elicitedboth T cell and antibody responses, with P501S alone eliciting CTLresponses but not antibody responses. Immunization with P501S alone wasfound to be most effective in combination with a P501S-adenovirus boost.Immunizations with P501S alone generated T cells that respond to peptide370 (CLSHSVAW). In contrast, immunizations with P501S-adenovirusgenerated weak or no reactivity to peptide 370. These studies indicatethat a combination of a DNA priming immunization followed by anadenovirus boost is a potent approach for eliciting both CTL andantibody response, and that distinct cytotoxic T cells, recognizingdifferent epitopes, may be elicited when immunizing with P501S or P501Splus adenovirus versus immunizing with adenovirus alone.

Example 8 Ability of Human T Cells to Recognize Prostate-SpecificPolypeptides

This Example illustrates the ability of T cells specific for a prostatetumor polypeptide to recognize human tumor.

Human CD8⁺ T cells were primed in vitro to the P2S-12 peptide (SEQ IDNO: 306) derived from P502S (also referred to as J1-17) using dendriticcells according to the protocol of Van Tsai et al. (Critical Reviews inImmunology 18:65-75, 1998). The resulting CD8⁺ T cell microcultures weretested for their ability to recognize the P2S-12 peptide presented byautologous fibroblasts or fibroblasts which were transduced to expressthe P502S gene in a γ-interferon ELISPOT assay (see Lalvani et al., J.Exp. Med. 186:859-865, 1997). Briefly, titrating numbers of T cells wereassayed in duplicate on 10⁴ fibroblasts in the presence of 3 μg/ml humanβ₂-microglobulin and 1 μg/ml P2S-12 peptide or control E75 peptide. Inaddition, T cells were simultaneously assayed on autologous fibroblaststransduced with the P502S gene or as a control, fibroblasts transducedwith HER-2/neu. Prior to the assay, the fibroblasts were treated with 10ng/ml γ-interferon for 48 hours to upregulate class I MHC expression.One of the microcultures (#5) demonstrated strong recognition of bothpeptide pulsed fibroblasts as well as transduced fibroblasts in aγ-interferon ELISPOT assay. FIG. 2A demonstrates that there was a strongincrease in the number of γ-interferon spots with increasing numbers ofT cells on fibroblasts pulsed with the P2S-12 peptide (solid bars) butnot with the control E75 peptide (open bars). This shows the ability ofthese T cells to specifically recognize the P2S-12 peptide. As shown inFIG. 2B, this microculture also demonstrated an increase in the numberof γ-interferon spots with increasing numbers of T cells on fibroblaststransduced to express the P502S gene but not the HER-2/neu gene. Theseresults provide additional confirmatory evidence that the P2S-12 peptideis a naturally processed epitope of the P502S protein. Furthermore, thisalso demonstrates that there exists in the human T cell repertoire, highaffinity T cells which are capable of recognizing this epitope. These Tcells should also be capable of recognizing human tumors which expressthe P502S gene.

Example 9 Elicitation of Prostate Antigen-Specific CTL Responses inHuman Blood and Identification of CTL Epitopes

This Example illustrates the ability of the prostate-specific antigenP501S to elicit a cytotoxic T lymphocyte (CTL) response in blood ofnormal humans and defines important epitopes responsible for T-cellrecognition. Such information further confirms the immunogenicity ofP501S and validates its use in any of a number of illustrativeimmunodiagnostic and immunotherapeutic embodiments described herein.

Autologous dendritic cells (DC) were differentiated from monocytecultures derived from PBMC of normal donors by growth for five days inRPMI medium containing 10% human serum, 50 ng/ml GMCSF and 30 ng/mlIL-4. Following culture, DC were infected overnight with recombinantP501S-expressing vaccinia virus at an M.O.I. of 5 and matured for 8hours by the addition of 2 micrograms/ml CD40 ligand. Virus wasinactivated by UV irradiation, CD8⁺ cells were isolated by positiveselection using magnetic beads, and priming cultures were initiated in24-well plates. Following five stimulation cycles using autologousfibroblasts retrovirally transduced to express P501S and CD80, CD8+lines were identified that specifically produced interferon-gamma whenstimulated with autologous P501S-transduced fibroblasts. TheP501S-specific activity of cell line 3A-1 could be maintained followingadditional stimulation cycles on autologous B-LCL transduced with P501S.Line 3A-1 was shown to specifically recognize autologous B-LCLtransduced to express P501S, but not EGFP-transduced autologous B-LCL,as measured by cytotoxicity assays (⁵¹Cr release) and interferon-gammaproduction (Interferon-gamma Elispot; see above and Lalvani et al., J.Exp. Med. 186:859-865, 1997). The results of these assays are presentedin FIGS. 6A and 6B.

The ability of the prostate antigen P501S to elicit an immune responsein human males was examined as follows.

Dendritic Cells (DC) were isolated by Percol gradient followed bydifferential adherence, and cultured for 5 days in the presence of RPMImedium containing 1% human serum, 50 ng/ml GM-CSF and 30 ng/ml IL-4.Following culture, DC were infected for 24 hours with P501S-expressingAdenovirus at an MOI of 10 and matured for an additional 24 hours by theaddition of 2 ug/ml CD40 ligand. CD8 cells were enriched from PBMC of anormal male donor by the subtraction of CD4+, CD14+ and CD16+populations from PBMC with magnetic beads. Priming cultures containing10,000 P501S-expressing DC and 100,000 CD8+ T cells per well were set upin 96-well V-bottom plates in RPMI containing 10% human serum, 5 ng/mlIL-12 and 10 ng/ml IL-6. Cultures were stimulated every 7 days usingautologous fibroblasts retrovirally transduced to express P501S andCD80, and were treated with IFN-gamma for 48-72 hours to upregulate MHCClass I expression. Ten u/ml IL-2 was added at the time of stimulationand on days 2 and 5 following stimulation. Following 4 stimulationcycles, multiple P501S-specific CD8+ T cell lines were identified thatproduced IFN-gamma in response to IFN-gamma treatedP501S/CD80-expressing autologous fibroblasts, but not in response toIFN-gamma treated P703P/CD80-expressing autologous fibroblasts in aγ-IFN Elispot assay. The three most promising lines, referred to as 1H1,2H2 and 6F1, were cloned in 96-well plates with 0.5 cell/well or 2cells/well in the presence of 75,000 PBMC/well, 10,000 B-LCL/well, 30ng/ml OKT3 and 50 u/ml IL-2 to isolate P501S-specific clones.

To identify the class I restriction element for the P501S-specificclones, HLA blocking and mismatch analyses were performed as follows. Inγ-IFN Elispot assays, the specific response of clones to P501Stransduced autologous fibroblasts was blocked by pre-incubation with 25ug/ml W6/32 (pan-Class I blocking antibody) and for 6F1 with BB7.2(HLA-A2 blocking antibody), and for 1H1 and 2H2 with B1.23.2 (HLA-B/Cblocking antibody). The results demonstrated that the P501S specificresponse of the clones is restricted to an HLA-A2 and/or HLA-B or -Callele. For the HLA mismatch analysis, autologous fibroblasts (D310,HLA-A2, A3, B44, B51, Cw5, Cw14) and heterologous fibroblasts (D326,HLA-A1, A2, B8, B51, Cw1, Cw7) that share the HLA-A-2 and HLAB51 allelewere transduced with P501S and used to stimulate the clones. In γ-IFNElispot assays, 1H1 and 2H2 only recognized the autologous fibroblasts,suggesting (together with the antibody blocking assays above) that therelevant allele was B44, Cw5, or Cw14, and 6F1 recognized both D310 andD326, suggesting the response was restricted to HLA-A0201.

To confirm and extend the above restriction data, each of the allelesfrom D310 was cloned into the pBIB expression vector and cotransfectedinto Cos-7 cells together with full-length P501S. 1H1 and 2H2 clonesspecifically recognized Cos-7 cells cotransfected with P501S and theCw0501 alelle, while 6F1 clones recognized Cos-7 cells cotransfectedwith P501S and the HLA-A0201 allele. These results demonstrate that theabove alleles are responsible for presenting P501S epitopes to the aboveCTL.

To identify a sub-fragment of P501S that contained the epitoperecognized by the CTL, 3′ truncations of P501S were cotransfected withthe above alleles into Cos-7 cells. A 6F1 clone was found to recognizean epitope derived from the initial 354 nucleotides of the P501S openreading frame (ORF), a 2H2 clone (referred to as 2H2-1A12) recognized anepitope derived from the sequence of P501S found between amino acids471-553 of P501S (SEQ ID NO: 113), and a 1H1 clone (referred to as1H1-1A6) recognized an epitope derived from the sequence of P501S foundbetween amino acids 278-353 of P501S (SEQ ID NO: 113).

To identify minimal epitopes recognized by these CTL, 15 or 19-merpeptides overlapping by 11 or 15 amino acids, respectively, weresynthesized, pulsed onto autologous B-LCL and used as antigen presentingcells for the CTL in γ-IFN ELISPOT assays. For 1H1-1A6, a pool of four15-21 mers that spanned amino acids 246-325 of SEQ ID NO: 113 wasspecifically recognized by the CTL. In the pool breakdown, peptide 291(amino acids 291-310; SEQ ID NO: 983, cDNA sequence provided in SEQ IDNO: 972) was specifically recognized by the 1H1-1A6 CTL. Ten-merpeptides from amino acids 291-310 of P501S were then synthesized andtested as above. Peptides 291, 292, and 293 (SEQ ID NO: 984, 982 and980, respectively; cDNA sequences provided in SEQ ID NO: 975, 976 and973, respectively) were all specifically recognized by the CTL. Theseresults demonstrate that each of the three 10-mer peptides contain theminimal epitope recognized by the CTL. The 8-mer peptide common to thethree 10-mers with the sequence TDFVGEGL (SEQ ID NO: 981) was thensynthesized and tested. In a peptide titration assay, the CD8+ T cellsspecific for this epitope recognized APC pulsed with 1-10 μg/ml. The8-mer was also recognized efficiently by the CTL, demonstrating that aminimal naturally processed peptide epitope recognized by the CTL spansSEQ ID NO: 981. Further analysis revealed that the minimal epitope forthe clone 1H1-1A6 was YTDFVGEGL (amino acids 292-300 of P501), the aminoacid and DNA sequences for which are disclosed in SEQ ID NOs:1021 and1022, respectively. Peptide titration assays revealed that CD8+ T cellsspecific for this epitope recognized APC pulsed with 0.0016 μg/ml of the9-mer. Therefore a minimal CD8+ T cell epitope specific for P501 isYTDFVGEGL (SEQ ID NO:1022).

For 2H2-1A12, a pool of twenty 15-mers that spanned amino acids 462-553was pulsed onto autologous B-LCL and tested as above. Peptides 462 and465 (SEQ ID NO: 979 and 977, respectively; cDNA sequences provided inSEQ ID NO: 971 and 969, respectively) were both specifically recognizedby the CTL. These results demonstrate that each of the two 15-merpeptides contain the minimal epitope recognized by the CTL. 10-merpeptides overlapping by a single amino acid were then synthesized fromthe sequence in common to the two above peptides and tested as above.Primarily peptide 464 (amino acids 464-474; SEQ ID NO: 978; cDNAsequence provided in SEQ ID NO: 970) and also peptides 463 (amino acids463-472) and 465 (amino acids 465-475) were specifically recognized bythe CTL. These results indicate that the sequence of SEQ ID NO: 978(amino acids 464-474 of P501S) contains the minimal epitope recognizedby 2H2-1A12 CTL. Further analysis revealed that the minimal epitope forthe clone 2H2-1A12 was ACDVSVRVV (amino acids 465-473 of P501S) theamino acid and DNA sequences of which are disclosed in SEQ ID NOs:1012and 1013, respectively. The recognition of peptides 463 and 465 eitherreflects contamination of these peptides with peptide 464, or additionalsecondary and inefficient recognition of these peptides by the CTL.

This example thus confirms that P501S is capable of eliciting a humanCTL response and identifies multiple CTL epitopes responsible for T-cellrecognition of this prostate-specific antigen. Such information furthervalidates P501S as an important target for use in immunodiagnostic andimmunotherapeutic applications.

Example 10 Identification of a Naturally Processed CTL Epitope Containedwithin the Prostate-Specific Antigen P703P

The 9-mer peptide p5 (SEQ ID NO: 338) was derived from the P703P antigen(also referred to as P20). The p5 peptide is immunogenic in human HLA-A2donors and is a naturally processed epitope. Antigen specific human CD8+T cells can be primed following repeated in vitro stimulations withmonocytes pulsed with p5 peptide. These CTL specifically recognizep5-pulsed and P703P-transduced target cells in both ELISPOT (asdescribed above) and chromium release assays. Additionally, immunizationof HLA-A2Kb transgenic mice with p5 leads to the generation of CTL lineswhich recognize a variety of HLA-A2Kb or HLA-A2 transduced target cellsexpressing P703P.

Initial studies demonstrating that p5 is a naturally processed epitopewere done using HLA-A2Kb transgenic mice. HLA-A2Kb transgenic mice wereimmunized subcutaneously in the footpad with 100 μg of p5 peptidetogether with 140 μg of hepatitis B virus core peptide (a Th peptide) inFreund's incomplete adjuvant. Three weeks post immunization, spleencells from immunized mice were stimulated in vitro with peptide-pulsedLPS blasts. CTL activity was assessed by chromium release assay fivedays after primary in vitro stimulation. Retrovirally transduced cellsexpressing the control antigen P703P and HLA-A2Kb were used as targets.CTL lines that specifically recognized both p5-pulsed targets as well asP703P-expressing targets were identified.

Human in vitro priming experiments demonstrated that the p5 peptide isimmunogenic in humans. Dendritic cells (DC) were differentiated frommonocyte cultures derived from PBMC of normal human donors by culturingfor five days in RPMI medium containing 10% human serum, 50 ng/ml humanGM-CSF and 30 ng/ml human IL-4. Following culture, the DC were pulsedwith 1 ug/ml p5 peptide and cultured with CD8+ T cell enriched PBMC. CTLlines were restimulated on a weekly basis with p5-pulsed monocytes. Fiveto six weeks after initiation of the CTL cultures, CTL recognition ofp5-pulsed target cells was demonstrated. CTL were additionally shown torecognize human cells transduced to express P703P, demonstrating that p5is a naturally processed epitope.

Studies identifying a further peptide epitope (referred to as peptide 4)derived from the prostate tumor-specific antigen P703P that is capableof being recognized by CD4 T cells on the surface of cells in thecontext of HLA class II molecules were carried out as follows. The aminoacid sequence for peptide 4 is provided in SEQ ID NO: 781, with thecorresponding cDNA sequence being provided in SEQ ID NO: 782.

Twenty 15-mer peptides overlapping by 10 amino acids and derived fromthe carboxy-terminal fragment of P703P were generated using standardprocedures. Dendritic cells (DC) were derived from PBMC of a normalfemale donor using GM-CSF and IL-4 by standard protocols. CD4 T cellswere generated from the same donor as the DC using MACS beads andnegative selection. DC were pulsed overnight with pools of the 15-merpeptides, with each peptide at a final concentration of 0.25microgram/ml. Pulsed DC were washed and plated at 1×10⁴ cells/well of96-well V-bottom plates and purified CD4 T cells were added at1×10⁵/well. Cultures were supplemented with 60 ng/ml IL-6 and 10 ng/mlIL-12 and incubated at 37° C. Cultures were restimulated as above on aweekly basis using DC generated and pulsed as above as antigenpresenting cells, supplemented with 5 ng/ml IL-7 and 10 u/ml IL-2.Following 4 in vitro stimulation cycles, 96 lines (each linecorresponding to one well) were tested for specific proliferation andcytokine production in response to the stimulating pools with anirrelevant pool of peptides derived from mammaglobin being used as acontrol.

One line (referred to as 1-F9) was identified from pool #1 thatdemonstrated specific proliferation (measured by 3H proliferationassays) and cytokine production (measured by interferon-gamma ELISAassays) in response to pool #1 of P703P peptides. This line was furthertested for specific recognition of the peptide pool, specificrecognition of individual peptides in the pool, and in HLA mismatchanalyses to identify the relevant restricting allele. Line 1-F9 wasfound to specifically proliferate and produce interferon-gamma inresponse to peptide pool #1, and also to peptide 4 (SEQ ID NO: 781).Peptide 4 corresponds to amino acids 126-140 of SEQ ID NO: 327. Peptidetitration experiments were conducted to assess the sensitivity of line1-F9 for the specific peptide. The line was found to specificallyrespond to peptide 4 at concentrations as low as 0.25 ng/ml, indicatingthat the T cells are very sensitive and therefore likely to have highaffinity for the epitope.

To determine the HLA restriction of the P703P response, a panel ofantigen presenting cells (APC) was generated that was partially matchedwith the donor used to generate the T cells. The APC were pulsed withthe peptide and used in proliferation and cytokine assays together withline 1-F9. APC matched with the donor at HLA-DRB0701 and HLA-DQB02alleles were able to present the peptide to the T cells, indicating thatthe P703P-specific response is restricted to one of these alleles.

Antibody blocking assays were utilized to determine if the restrictingallele was HLA-DR0701 or HLA-DQ02. The anti-HLA-DR blocking antibodyL243 or an irrelevant isotype matched IgG2a were added to T cells andAPC cultures pulsed with the peptide RMPTVLQCVNVSVVS (SEQ ID NO: 781) at250 ng/ml. Standard interferon-gamma and proliferation assays wereperformed. Whereas the control antibody had no effect on the ability ofthe T cells to recognize peptide-pulsed APC, in both assays theanti-HLA-DR antibody completely blocked the ability of the T cells tospecifically recognize peptide-pulsed APC.

To determine if the peptide epitope RMPTVLQCVNVSVVS (SEQ ID NO: 781) wasnaturally processed, the ability of line 1-F9 to recognize APC pulsedwith recombinant P703P protein was examined. For these experiments anumber of recombinant P703P sources were utilized; E. coli-derivedP703P, Pichia-derived P703P and baculovirus-derived P703P. Irrelevantprotein controls used were E. coli-derived L3E a lung-specific antigen)and baculovirus-derived mammaglobin. In interferon-gamma ELISA assays,line 1-F9 was able to efficiently recognize both E. coli forms of P703Pas well as Pichia-derived recombinant P703P, while baculovirus-derivedP703P was recognized less efficiently. Subsequent Western blot analysisrevealed that the E coli and Pichia P703P protein preparations wereintact while the baculovirus P703P preparation was approximately 75%degraded. Thus, peptide RMPTVLQCVNVSVVS (SEQ ID NO: 781) from P703P is anaturally processed peptide epitope derived from P703P and presented toT cells in the context of HLA-DRB-0701

In further studies, twenty-four 15-mer peptides overlapping by 10 aminoacids and derived from the N-terminal fragment of P703P (correspondingto amino acids 27-154 of SEQ ID NO: 525) were generated by standardprocedures and their ability to be recognized by CD4 cells wasdetermined essentially as described above. DC were pulsed overnight withpools of the peptides with each peptide at a final concentration of 10microgram/ml. A large number of individual CD4 T cell lines (65/480)demonstrated significant proliferation and cytokine release (IFN-gamma)in response to the P703P peptide pools but not to a control peptidepool. The CD4 T cell lines which demonstrated specific activity wererestimulated on the appropriate pool of P703P peptides and reassayed onthe individual peptides of each pool as well as a peptide dose titrationof the pool of peptides in a IFN-gamma release assay and in aproliferation assay.

Sixteen immunogenic peptides were recognized by the T cells from theentire set of peptide antigens tested. The amino acid sequences of thesepeptides are provided in SEQ ID NO: 799-814, with the corresponding cDNAsequences being provided in SEQ ID NO: 783-798, respectively. In somecases the peptide reactivity of the T cell line could be mapped to asingle peptide, however some could be mapped to more than one peptide ineach pool. Those CD4 T cell lines that displayed a representativepattern of recognition from each peptide pool with a reasonable affinityfor peptide were chosen for further analysis (I-1A, -6A; II-4C, -5E;III-6E, IV-4B, -3F, -9B, -10F, V-5B, -4D, and -10F). These CD4 T cellslines were restimulated on the appropriate individual peptide andreassayed on autologous DC pulsed with a truncated form of recombinantP703P protein made in E. coli (a.a. 96-254 of SEQ ID NO: 525),full-length P703P made in the baculovirus expression system, and afusion between influenza virus NS1 and P703P made in E. coli. Of the Tcell lines tested, line I-1A recognized specifically the truncated formof P703P (E. coli) but no other recombinant form of P703P. This linealso recognized the peptide used to elicit the T cells. Line 2-4Crecognized the truncated form of P703P (E. coli) and the full lengthform of P703P made in baculovirus, as well as peptide. The remaining Tcell lines tested were either peptide-specific only (II-5E, II-6F,IV-4B, IV-3F, IV-9B, IV-10F, V-5B and V-4D) or were non-responsive toany antigen tested (V-10F). These results demonstrate that the peptidesequence RPLLANDLMLIKLDE (SEQ ID NO: 814; corresponding to a.a. 110-124of SEQ ID NO: 525) recognized by the T cell line I-1A, and the peptidesequences SVSESDTIRSISIAS (SEQ ID NO: 811; corresponding to a.a. 125-139of SEQ ID NO: 525) and ISIASQCPTAGNSCL (SEQ ID NO: 810; corresponding toa.a. 135-149 of SEQ ID NO: 525) recognized by the T cell line II-4C maybe naturally processed epitopes of the P703P protein.

In further studies, forty 15-mer peptides overlapping by 10 amino acidsand derived spanning amino acids 47 to 254 of P703P (SEQ ID NO: 525)were generated by standard procedures and their ability to be recognizedby CD4 cells was determined essentially as described above. DC wereprepared from PBMC of a donor having distinct HLA DR and DQ alleles fromthat used in previous experiments. DC were pulsed overnight with poolsof the peptides with each peptide at a final concentration of 0.25microgram/ml, and each pool containing 10 peptides. Twelve lines wereidentified that demonstrated specific proliferation and cytokineproduction (measured in gamma-interferon ELISA assays) in response tothe stimulating peptide pool. These lines were further tested forspecific recognition of the peptide pool, specific recognition ofindividual peptides in the pool, and specific recognition of recombinantP703P protein. Lines 3A5H and 3A9H specifically proliferated andproduced gamma-interferon in response to recombinant protein and oneindividual peptide as well as the peptide pool. Following re-stimulationon targets loaded with the specific peptide, only 3A9H respondedspecifically to targets exposed to lysates of fibroblasts infectedadenovirus expressing full-length P703P. These results indicates thatthe line 3A9H can respond to antigenic peptide derived from proteinsynthesized in mammalian cells. The peptide to which the specific CD4line responded correspond to amino acids 155-170 of P703P (SEQ ID NO:943). The DNA sequence for this peptide is provided in SEQ ID NO: 942.

Example 11 Expression of a Breast Tumor-Derived Antigen in Prostate

Isolation of the antigen B305D from breast tumor by differential displayis described in U.S. patent application Ser. No. 08/700,014, filed Aug.20, 1996. Several different splice forms of this antigen were isolated.The determined cDNA sequences for these splice forms are provided in SEQID NO: 366-375, with the amino acid sequences corresponding to thesequences of SEQ ID NO: 292, 298 and 301-303 being provided in SEQ IDNO: 299-306, respectively. In further studies, a splice variant of thecDNA sequence of SEQ ID NO: 366 was isolated which was found to containan additional guanine residue at position 884 (SEQ ID NO: 530), leadingto a frameshift in the open reading frame. The determined DNA sequenceof this ORF is provided in SEQ ID NO: 531. This frameshift generates aprotein sequence (provided in SEQ ID NO: 532) of 293 amino acids thatcontains the C-terminal domain common to the other isoforms of B305D butthat differs in the N-terminal region.

The expression levels of B305D in a variety of tumor and normal tissueswere examined by real time PCR and by Northern analysis. The resultsindicated that B305D is highly expressed in breast tumor, prostatetumor, normal prostate and normal testes, with expression being low orundetectable in all other tissues examined (colon tumor, lung tumor,ovary tumor, and normal bone marrow, colon, kidney, liver, lung, ovary,skin, small intestine, stomach). Using real-time PCR on a panel ofprostate tumors, expression of B305D in prostate tumors was shown toincrease with increasing Gleason grade, demonstrating that expression ofB305D increases as prostate cancer progresses.

Example 12 Generation of Human CTL In Vitro Using Whole Gene Priming andStimulation Techniques with the Prostate-Specific Antigen P501S

Using in vitro whole-gene priming with P501S-vaccinia infected DC (see,for example, Yee et al, The Journal of Immunology, 157(9):4079-86,1996), human CTL lines were derived that specifically recognizeautologous fibroblasts transduced with P501S (also known as L1-12), asdetermined by interferon-γ ELISPOT analysis as described above. Using apanel of HLA-mismatched B-LCL lines transduced with P501S, these CTLlines were shown to be likely restricted to HLAB class I allele.Specifically, dendritic cells (DC) were differentiated from monocytecultures derived from PBMC of normal human donors by growing for fivedays in RPMI medium containing 10% human serum, 50 ng/ml human GM-CSFand 30 ng/ml human IL-4. Following culture, DC were infected overnightwith recombinant P501S vaccinia virus at a multiplicity of infection(M.O.I) of five, and matured overnight by the addition of 3 μg/ml CD40ligand. Virus was inactivated by UV irradiation. CD8+ T cells wereisolated using a magnetic bead system, and priming cultures wereinitiated using standard culture techniques. Cultures were restimulatedevery 7-10 days using autologous primary fibroblasts retrovirallytransduced with P501S and CD80. Following four stimulation cycles, CD8+T cell lines were identified that specifically produced interferon-γwhen stimulated with P501S and CD80-transduced autologous fibroblasts. Apanel of HLA-mismatched B-LCL lines transduced with P501S were generatedto define the restriction allele of the response. By measuringinterferon-γ in an ELISPOT assay, the P501S specific response was shownto be likely restricted by HLA B alleles. These results demonstrate thata CD8+ CTL response to P501S can be elicited.

To identify the epitope(s) recognized, cDNA encoding P501S wasfragmented by various restriction digests, and sub-cloned into theretroviral expression vector pBIB-KS. Retroviral supernatants weregenerated by transfection of the helper packaging line Phoenix-Ampho.Supernatants were then used to transduce Jurkat/A2Kb cells for CTLscreening. CTL were screened in IFN-gamma ELISPOT assays against theseA2Kb targets transduced with the “library” of P501S fragments. Initialpositive fragments P501S/H3 and P501S/F2 were sequenced and found toencode amino acids 106-553 and amino acids 136-547, respectively, of SEQID NO: 113. A truncation of H3 was made to encode amino acid residues106-351 of SEQ ID NO: 113, which was unable to stimulate the CTL, thuslocalizing the epitope to amino acid residues 351-547. Additionalfragments encoding amino acids 1-472 (Fragment A) and amino acids 1-351(Fragment B) were also constructed. Fragment A but not Fragment Bstimulated the CTL thus localizing the epitope to amino acid residues351-472. Overlapping 20-mer and 18-mer peptides representing this regionwere tested by pulsing Jurkat/A2Kb cells versus CTL in an IFN-gammaassay. Only peptides P501S-369(20) and P501S-369(18) stimulated the CTL.Nine-mer and 10-mer peptides representing this region were synthesizedand similarly tested. Peptide P501S-370 (SEQ ID NO: 539) was the minimal9-mer giving a strong response. Peptide P501S-376 (SEQ ID NO: 540) alsogave a weak response, suggesting that it might represent across-reactive epitope.

In subsequent studies, the ability of primary human B cells transducedwith P501S to prime MHC class I-restricted, P501S-specific, autologousCD8 T cells was examined. Primary B cells were derived from PBMC of ahomozygous HLA-A2 donor by culture in CD40 ligand and IL-4, transducedat high frequency with recombinant P501S in the vector pBIB, andselected with blastocidin-S. For in vitro priming, purified CD8+ T cellswere cultured with autologous CD40 ligand+IL-4 derived, P501S-transducedB cells in a 96-well microculture format. These CTL microcultures werere-stimulated with P501S-transduced B cells and then assayed forspecificity. Following this initial screen, microcultures withsignificant signal above background were cloned on autologousEBV-transformed B cells (BLCL), also transduced with P501S. UsingIFN-gamma ELISPOT for detection, several of these CD8 T cell clones werefound to be specific for P501S, as demonstrated by reactivity toBLCL/P501S but not BLCL transduced with control antigen. It was furtherdemonstrated that the anti-P501S CD8 T cell specificity isHLA-A2-restricted. First, antibody blocking experiments withanti-HLA-A,B,C monoclonal antibody (W6.32), anti-HLA-B,C monoclonalantibody (B1.23.2) and a control monoclonal antibody showed that onlythe anti-HLA-A,B,C antibody blocked recognition of P501S-expressingautologous BLCL. Secondly, the anti-P501S CTL also recognized an HLA-A2matched, heterologous BLCL transduced with P501S, but not thecorresponding EGFP transduced control BLCL.

A naturally processed, CD8, class I-restricted peptide epitope of P501Swas identified as follows. Dendritic cells (DC) were isolated by Percolgradient followed by differential adherence, and cultured for 5 days inthe presence of RPMI medium containing 1% human serum, 50 ng/ml GM-CSFand 30 ng/ml IL-4. Following culture, DC were infected for 24 hours withP501S-expressing adenovirus at an MOI of 10 and matured for anadditional 24 hours by the addition of 2 ug/ml CD40 ligand. CD8 cellswere enriched for by the subtraction of CD4+, CD14+ and CD16+populations from PBMC with magnetic beads. Priming cultures containing10,000 P501S-expressing DC and 100,000 CD8+ T cells per well were set upin 96-well V-bottom plates with RPMI containing 10% human serum, 5 ng/mlIL-12 and 10 ng/ml IL-6. Cultures were stimulated every 7 days usingautologous fibroblasts retrovirally transduced to express P501S andCD80, and were treated with IFN-gamma for 48-72 hours to upregulate MHCClass I expression. 10 u/ml IL-2 was added at the time of stimulationand on days 2 and 5 following stimulation. Following 4 stimulationcycles, one P501S-specific CD8+ T cell line (referred to as 2A2) wasidentified that produced IFN-gamma in response to IFN-gamma-treatedP501S/CD80 expressing autologous fibroblasts, but not in response toIFN-gamma-treated P703P/CD80 expressing autologous fibroblasts in aγ-IFN Elispot assay. Line 2A2 was cloned in 96-well plates with 0.5cell/well or 2 cells/well in the presence of 75,000 PBMC/well, 10,000B-LCL/well, 30 ng/ml OKT3 and 50u/ml IL-2. Twelve clones were isolatedthat showed strong P501S specificity in response to transducedfibroblasts.

Fluorescence activated cell sorting (FACS) analysis was performed onP501S-specific clones using CD3-, CD4- and CD8-specific antibodiesconjugated to PercP, FITC and PE respectively. Consistent with the useof CD8 enriched T cells in the priming cultures, P5401S-specific cloneswere determined to be CD3+, CD8+ and CD4-.

To identify the relevant P501S epitope recognized by P501S specific CTL,pools of 18-20 mer or 30-mer peptides that spanned the majority of theamino acid sequence of P501S were loaded onto autologous B-LCL andtested in γ-IFN Elispot assays for the ability to stimulate twoP501S-specific CTL clones, referred to as 4E5 and 4E7. One pool,composed of five 18-20 mer peptides that spanned amino acids 411-486 ofP501S (SEQ ID NO: 113), was found to be recognized by bothP501S-specific clones. To identify the specific 18-20 mer peptiderecognized by the clones, each of the 18-20 mer peptides that comprisedthe positive pool were tested individually in γ-IFN Elispot assays forthe ability to stimulate the two P501S-specific CTL clones, 4E5 and 4E7.Both 4E5 and 4E7 specifically recognized one 20-mer peptide (SEQ ID NO:853; cDNA sequence provided in SEQ ID NO: 854) that spanned amino acids453-472 of P501S. Since the minimal epitope recognized by CD8+ T cellsis almost always either a 9 or 10-mer peptide sequence, 10-mer peptidesthat spanned the entire sequence of SEQ ID NO: 853 were synthesized thatdiffered by 1 amino acid. Each of these 10-mer peptides was tested forthe ability to stimulate two P501S-specific clones, (referred to as 1D5and 1E12). One 10-mer peptide (SEQ ID NO: 855; cDNA sequence provided inSEQ ID NO: 856) was identified that specifically stimulated theP501S-specific clones. This epitope spans amino acids 463-472 of P501S.This sequence defines a minimal 10-mer epitope from P501S that can benaturally processed and to which CTL responses can be identified innormal PBMC. Thus, this epitope is a candidate for use as a vaccinemoiety, and as a therapeutic and/or diagnostic reagent for prostatecancer.

To identify the class I restriction element for the P501S-derivedsequence of SEQ ID NO: 855, HLA blocking and mismatch analyses wereperformed. In γ-IFN Elispot assays, the specific response of clones 4A7and 4E5 to P501S-transduced autologous fibroblasts was blocked bypre-incubation with 25 ug/ml W6/32 (pan-Class I blocking antibody) andB1.23.2 (HLA-B/C blocking antibody). These results demonstrate that theSEQ ID NO: 855-specific response is restricted to an HLA-B or HLA-Callele.

For the HLA mismatch analysis, autologous B-LCL (HLA-A1, A2, B8, B51,Cw1, Cw7) and heterologous B-LCL (HLA-A2, A3, B18, B51, Cw5, Cw14) thatshare the HLAB51 allele were pulsed for one hour with 20 ug/ml ofpeptide of SEQ ID NO: 855, washed, and tested in γ-IFN Elispot assaysfor the ability to stimulate clones 4A7 and 4E5. Antibody blockingassays with the B1.23.2 (HLA-B/C blocking antibody) were also performed.SEQ ID NO: 855-specific response was detected using both the autologous(D326) and heterologous (D107) B-LCL, and furthermore the responses wereblocked by pre-incubation with 25 ug/ml of B1.23.2 HLA-B/C blockingantibody. Together these results demonstrate that the P501S-specificresponse to the peptide of SEQ ID NO: 855 is restricted to the HLA-B51class I allele. Molecular cloning and sequence analysis of the HLA-B51allele from D326 revealed that the HLA-B51 subtype of D326 isHLA-B51011.

Based on the 10-mer P501S-derived epitope of SEQ ID NO: 855, two 9-merswith the sequences of SEQ ID NO: 857 and 858 were synthesized and testedin Elispot assays for the ability to stimulate two P501S-specific CTLclones derived from line 2A2. The 10-mer peptide of SEQ ID NO: 855, aswell as the 9-mer peptide of SEQ ID NO: 858, but not the 9-mer peptideof SEQ ID NO: 857, were capable of stimulating the P501S-specific CTL toproduce IFN-gamma. These results demonstrate that the peptide of SEQ IDNO: 858 is a 9-mer P501S-derived epitope recognized by P501S-specificCTL. The DNA sequence encoding the epitope of SEQ ID NO: 858 is providedin SEQ ID NO: 859.

To identify the class I restricting allele for the P501S-derived peptideof SEQ ID NO: 855 and 858 specific response, each of the HLA B and Calleles were cloned from the donor used in the in vitro primingexperiment. Sequence analysis indicated that the relevant alleles wereHLA-B8, HLA-B51, HLA-Cw01 and HLA-Cw07. Each of these alleles weresubcloned into an expression vector and co-transfected together with theP501S gene into VA-13 cells. Transfected VA-13 cells were then testedfor the ability to specifically stimulate the P501S-specific CTL inELISPOT assays. VA-13 cells transfected with P501S and HLA-B51 werecapable of stimulating the P501S-specific CTL to secrete gamma-IFN.VA-13 cells transfected with HLA-B51 alone or P501S+ the otherHLA-alleles were not capable of stimulating the P501S-specific CTL.These results demonstrate that the restricting allele for theP501S-specific response is the HLAB51 allele. Sequence analysis revealedthat the subtype of the relevant restricting allele is HLA-B51011.

To determine if the P501S-specific CTL could recognize prostate tumorcells that express P501S, the P501S-positive lines LnCAP and CRL2422(both expressing “moderate” amounts of P501S mRNA and protein), and PC-3(expressing low amounts of P501S mRNA and protein), plus theP501S-negative cell line DU-145 were retrovirally transduced with theHLA-B51011 allele that was cloned from the donor used to generate theP501S-specific CTL. HLA-B51011- or EGFP-transduced and selected tumorcells were treated with gamma-interferon and androgen (to upregulatestimulatory functions and P501S, respectively) and used ingamma-interferon Elispot assays with the P501S-specific CTL clones 4E5and 4E7. Untreated cells were used as a control.

Both 4E5 and 4E7 efficiently and specifically recognized LnCAP andCRL2422 cells that were transduced with the HLA-B51011 allele, but notthe same cell lines transduced with EGFP. Additionally, both CTL clonesspecifically recognized PC-3 cells transduced with HLA-B51011, but notthe P501S-negative tumor cell line DU-145. Treatment withgamma-interferon or androgen did not enhance the ability of CTL torecognize tumor cells. These results demonstrate that P501S-specificCTL, generated by in vitro whole gene priming, specifically andefficiently recognize prostate tumor cell lines that express P501S.

A naturally processed CD4 epitope of P501S was identified as follows.

CD4 cells specific for P501S were prepared as described above. A seriesof 16 overlapping peptides were synthesized that spanned approximately50% of the amino terminal portion of the P501S gene (amino acids 1-325of SEQ ID NO: 113). For priming, peptides were combined into pools of 4peptides, pulsed at 4 μg/ml onto dendritic cells (DC) for 24 hours, withTNF-alpha. DC were then washed and mixed with negatively selected CD4+ Tcells in 96 well U-bottom plates. Cultures were re-stimulated weekly onfresh DC loaded with peptide pools. Following a total of 4 stimulationcycles, cells were rested for an additional week and tested forspecificity to APC pulsed with peptide pools using γ-IFN ELISA andproliferation assays. For these assays, adherent monocytes loaded witheither the relevant peptide pool at 4 ug/ml or an irrelevant peptide atμg/ml were used as APC. T cell lines that demonstrated either specificcytokine secretion or proliferation were then tested for recognition ofindividual peptides that were present in the pool. T cell lines could beidentified from pools A and B that recognized individual peptides fromthese pools.

From pool A, lines AD9 and AE10 specifically recognized peptide 1 (SEQID NO: 862), and line AF5 recognized peptide 39 (SEQ ID NO: 861). Frompool B, line BC6 could be identified that recognized peptide 58 (SEQ IDNO: 860). Each of these lines were stimulated on the specific peptideand tested for specific recognition of the peptide in a titration assayas well as cell lysates generated by infection of HEK 293 cells withadenovirus expressing either P501S or an irrelevant antigen. For theseassays, APC-adherent monocytes were pulsed with either 10, 1, or 0.1μg/ml individual P501S peptides, and DC were pulsed overnight with a 1:5dilution of adenovirally infected cell lysates. Lines AD9, AE10 and AF5retained significant recognition of the relevant P501S-derived peptideseven at 0.1 mg/ml. Furthermore, line AD9 demonstrated significant (8.1fold stimulation index) specific activity for lysates fromadenovirus-P501S infected cells. These results demonstrate that highaffinity CD4 T cell lines can be generated toward P501S-derivedepitopes, and that at least a subset of these T cells specific for theP501S derived sequence of SEQ ID NO: 862 are specific for an epitopethat is naturally processed by human cells. The DNA sequences encodingthe amino acid sequences of SEQ ID NO: 860-862 are provided in SEQ IDNO: 863-865, respectively.

To further characterize the P501S-specific activity of AD9, the line wascloned using anti-CD3. Three clones, referred to as 1A1, 1A9 and 1F5,were identified that were specific for the P501S-1 peptide (SEQ ID NO:862). To determine the HLA restriction allele for the P501S-specificresponse, each of these clones was tested in class II antibody blockingand HLA mismatch assays using proliferation and gamma-interferon assays.In antibody blocking assays and measuring gamma-interferon productionusing ELISA assays, the ability of all three clones to recognize peptidepulsed APC was specifically blocked by co-incubation with either apan-class II blocking antibody or a HLA-DR blocking antibody, but notwith a HLA-DQ or an irrelevant antibody. Proliferation assays performedsimultaneously with the same cells confirmed these results. These dataindicate that the P501S-specific response of the clones is restricted byan HLA-DR allele. Further studies demonstrated that the restrictingallele for the P501S-specific response is HLA-DRB1501.

Example 13 Identification of Prostate-Specific Antigens by MicroarrayAnalysis

This Example describes the isolation of certain prostate-specificpolypeptides from a prostate tumor cDNA library.

A human prostate tumor cDNA expression library as described above wasscreened using microarray analysis to identify clones that display atleast a three fold over-expression in prostate tumor and/or normalprostate tissue, as compared to non-prostate normal tissues (notincluding testis). 372 clones were identified, and 319 were successfullysequenced. Table II presents a summary of these clones, which are shownin SEQ ID NO:385-400. Of these sequences SEQ ID NO:386, 389, 390 and 392correspond to novel genes, and SEQ ID NO: 393 and 396 correspond topreviously identified sequences. The others (SEQ ID NO:385, 387, 388,391, 394, 395 and 397-400) correspond to known sequences, as shown inTable II.

TABLE II SUMMARY OF PROSTATE TUMOR ANTIGENS Previously Identified KnownGenes Genes Novel Genes T-cell gamma chain P504S 23379 (SEQ ID NO: 389)Kallikrein P1000C 23399 (SEQ ID NO: 392) Vector P501S 23320 (SEQ ID NO:386) CGI-82 protein mRNA (23319; P503S 23381 (SEQ ID SEQ ID NO: 385) NO:390) PSA P510S Ald. 6 Dehyd. P784P L-iditol-2 dehydrogenase (23376;P502S SEQ ID NO: 388) Ets transcription factor PDEF (22672; P706P SEQ IDNO: 398) hTGR (22678; SEQ ID NO: 399) 19142.2, bangur.seq (22621; SEQ IDNO: 396) KIAA0295(22685; SEQ ID NO: 400) 5566.1 Wang (23404; SEQ ID NO:393) Prostatic Acid Phosphatase(22655; P712P SEQ ID NO: 397)transglutaminase (22611; SEQ ID P778P NO: 395) HDLBP (23508; SEQ ID NO:394) CGI-69 Protein(23367; SEQ ID NO: 387) KIAA0122(23383; SEQ ID NO:391) TEEG

Subsequent studies led to the isolation of an extended cDNA sequence forthe clone of SEQ ID NO: 329 (also referred to as P554S). This extendedsequence is provided in SEQ ID NO: 967, with the corresponding aminoacid sequence being provided in SEQ ID NO: 968.

CGI-82 showed 4.06 fold over-expression in prostate tissues as comparedto other normal tissues tested. It was over-expressed in 43% of prostatetumors, 25% normal prostate, not detected in other normal tissuestested. L-iditol-2 dehydrogenase showed 4.94 fold over-expression inprostate tissues as compared to other normal tissues tested. It wasover-expressed in 90% of prostate tumors, 100% of normal prostate, andnot detected in other normal tissues tested. Ets transcription factorPDEF showed 5.55 fold over-expression in prostate tissues as compared toother normal tissues tested. It was over-expressed in 47% prostatetumors, 25% normal prostate and not detected in other normal tissuestested. hTGR1 showed 9.11 fold over-expression in prostate tissues ascompared to other normal tissues tested. It was over-expressed in 63% ofprostate tumors and is not detected in normal tissues tested includingnormal prostate. KIAA0295 showed 5.59 fold over-expression in prostatetissues as compared to other normal tissues tested. It wasover-expressed in 47% of prostate tumors, low to undetectable in normaltissues tested including normal prostate tissues. Prostatic acidphosphatase showed 9.14 fold over-expression in prostate tissues ascompared to other normal tissues tested. It was over-expressed in 67% ofprostate tumors, 50% of normal prostate, and not detected in othernormal tissues tested. Transglutaminase showed 14.84 foldover-expression in prostate tissues as compared to other normal tissuestested. It was over-expressed in 30% of prostate tumors, 50% of normalprostate, and is not detected in other normal tissues tested. Highdensity lipoprotein binding protein (HDLBP) showed 28.06 foldover-expression in prostate tissues as compared to other normal tissuestested. It was over-expressed in 97% of prostate tumors, 75% of normalprostate, and is undetectable in all other normal tissues tested. CGI-69showed 3.56 fold over-expression in prostate tissues as compared toother normal tissues tested. It is a low abundant gene, detected in morethan 90% of prostate tumors, and in 75% normal prostate tissues. Theexpression of this gene in normal tissues was very low. KIAA0122 showed4.24 fold over-expression in prostate tissues as compared to othernormal tissues tested. It was over-expressed in 57% of prostate tumors,it was undetectable in all normal tissues tested including normalprostate tissues. 19142.2 bangur showed 23.25 fold over-expression inprostate tissues as compared to other normal tissues tested. It wasover-expressed in 97% of prostate tumors and 100% of normal prostate. Itwas undetectable in other normal tissues tested. 5566.1 Wang showed 3.31fold over-expression in prostate tissues as compared to other normaltissues tested. It was over-expressed in 97% of prostate tumors, 75%normal prostate and was also over-expressed in normal bone marrow,pancreas, and activated PBMC. Novel clone 23379 (also referred to asP553S) showed 4.86 fold over-expression in prostate tissues as comparedto other normal tissues tested. It was detectable in 97% of prostatetumors and 75% normal prostate and is undetectable in all other normaltissues tested. Novel clone 23399 showed 4.09 fold over-expression inprostate tissues as compared to other normal tissues tested. It wasover-expressed in 27% of prostate tumors and was undetectable in allnormal tissues tested including normal prostate tissues. Novel clone23320 showed 3.15 fold over-expression in prostate tissues as comparedto other normal tissues tested. It was detectable in all prostate tumorsand 50% of normal prostate tissues. It was also expressed in normalcolon and trachea. Other normal tissues do not express this gene at highlevel.

Subsequent full-length cloning studies on P553S, using standardtechniques, revealed that this clone is an incomplete spliced form ofP501S. The determined cDNA sequences for four splice variants of P553Sare provided in SEQ ID NO: 702-705. An amino acid sequence encoded bySEQ ID NO: 705 is provided in SEQ ID NO: 706. The cDNA sequence of SEQID NO: 702 was found to contain two open reading frames (ORFs). Theamino acid sequences encoded by these two ORFs are provided in SEQ IDNO: 707 and 708.

Example 14 Identification of Prostate-Specific Antigens by ElectronicSubtraction

This Example describes the use of an electronic subtraction technique toidentify prostate-specific antigens.

Potential prostate-specific genes present in the GenBank human ESTdatabase were identified by electronic subtraction (similar to thatdescribed by Vasmatizis et al., Proc. Nat'l Acad. Sci. USA 95:300-304,1998). The sequences of EST clones (43,482) derived from variousprostate libraries were obtained from the GenBank public human ESTdatabase. Each prostate EST sequence was used as a query sequence in aBLASTN (National Center for Biotechnology Information) search againstthe human EST database. All matches considered identical (length ofmatching sequence >100 base pairs, density of identical matches overthis region >70%) were grouped (aligned) together in a cluster. Clusterscontaining more than 200 ESTs were discarded since they probablyrepresented repetitive elements or highly expressed genes such as thosefor ribosomal proteins. If two or more clusters shared common ESTs,those clusters were grouped together into a “supercluster,” resulting in4,345 prostate superclusters.

Records for the 479 human cDNA libraries represented in the GenBankrelease were downloaded to create a database of these cDNA libraryrecords. These 479 cDNA libraries were grouped into three groups: Plus(normal prostate and prostate tumor libraries, and breast cell linelibraries, in which expression was desired), Minus (libraries from othernormal adult tissues, in which expression was not desirable), and Other(libraries from fetal tissue, infant tissue, tissues found only inwomen, non-prostate tumors and cell lines other than prostate celllines, in which expression was considered to be irrelevant). A summaryof these library groups is presented in Table III.

TABLE III PROSTATE cDNA LIBRARIES AND ESTs Library # of Libraries # ofESTs Plus 25 43,482 Normal 11 18,875 Tumor 11 21,769 Cell lines 3 2,838Minus 166 Other 287

Each supercluster was analyzed in terms of the ESTs within thesupercluster. The tissue source of each EST clone was noted and used toclassify the superclusters into four groups: Type 1-EST clones found inthe Plus group libraries only; no expression detected in Minus or Othergroup libraries; Type 2-EST clones derived from the Plus and Other grouplibraries only; no expression detected in the Minus group; Type 3-ESTclones derived from the Plus, Minus and Other group libraries, but thenumber of ESTs derived from the Plus group is higher than in either theMinus or Other groups; and Type 4-EST clones derived from Plus, Minusand Other group libraries, but the number derived from the Plus group ishigher than the number derived from the Minus group. This analysisidentified 4,345 breast clusters (see Table IV). From these clusters,3,172 EST clones were ordered from Research Genetics, Inc., and werereceived as frozen glycerol stocks in 96-well plates.

TABLE IV PROSTATE CLUSTER SUMMARY # of # of ESTs Type SuperclustersOrdered 1 688 677 2 2899 2484 3 85 11 4 673 0 Total 4345 3172

The EST clone inserts were PCR-amplified using amino-linked PCR primersfor Synteni microarray analysis. When more than one PCR product wasobtained for a particular clone, that PCR product was not used forexpression analysis. In total, 2,528 clones from the electronicsubtraction method were analyzed by microarray analysis to identifyelectronic subtraction breast clones that had high levels of tumor vs.normal tissue mRNA. Such screens were performed using a Synteni (PaloAlto, Calif.) microarray, according to the manufacturer's instructions(and essentially as described by Schena et al., Proc. Nat'l Acad. Sci.USA 93:10614-10619, 1996 and Heller et al., Proc. Nat'l Acad. Sci. USA94:2150-2155, 1997). Within these analyses, the clones were arrayed onthe chip, which was then probed with fluorescent probes generated fromnormal and tumor prostate cDNA, as well as various other normal tissues.The slides were scanned and the fluorescence intensity was measured.

Clones with an expression ratio greater than 3 (i.e., the level inprostate tumor and normal prostate mRNA was at least three times thelevel in other normal tissue mRNA) were identified as prostatetumor-specific sequences (Table V). The sequences of these clones areprovided in SEQ ID NO: 401-453, with certain novel sequences shown inSEQ ID NO: 407, 413, 416-419, 422, 426, 427 and 450.

TABLE V PROSTATE-TUMOR SPECIFIC CLONES Sequence SEQ ID NO. DesignationComments 401 22545 previously identified P1000C 402 22547 previouslyidentified P704P 403 22548 known 404 22550 known 405 22551 PSA 406 22552prostate secretory protein 94 407 22553 novel 408 22558 previouslyidentified P509S 409 22562 glandular kallikrein 410 22565 previouslyidentified P1000C 411 22567 PAP 412 22568 B1006C (breast tumor antigen)413 22570 novel 414 22571 PSA 415 22572 previously identified P706P 41622573 novel 417 22574 novel 418 22575 novel 419 22580 novel 420 22581PAP 421 22582 prostatic secretory protein 94 422 22583 novel 423 22584prostatic secretory protein 94 424 22585 prostatic secretory protein 94425 22586 known 426 22587 novel 427 22588 novel 428 22589 PAP 429 22590known 430 22591 PSA 431 22592 known 432 22593 Previously identifiedP777P 433 22594 T cell receptor gamma chain 434 22595 Previouslyidentified P705P 435 22596 Previously identified P707P 436 22847 PAP 43722848 known 438 22849 prostatic secretory protein 57 439 22851 PAP 44022852 PAP 441 22853 PAP 442 22854 previously identified P509S 443 22855previously identified P705P 444 22856 previously identified P774P 44522857 PSA 446 23601 previously identified P777P 447 23602 PSA 448 23605PSA 449 23606 PSA 450 23612 novel 451 23614 PSA 452 23618 previouslyidentified P1000C 453 23622 previously identified P705P

Further studies on the clone of SEQ ID NO: 407 (also referred to asP1020C) led to the isolation of an extended cDNA sequence provided inSEQ ID NO: 591. This extended cDNA sequence was found to contain an openreading frame that encodes the amino acid sequence of SEQ ID NO: 592.The P1020C cDNA and amino acid sequences were found to show somesimilarity to the human endogenous retroviral HERV-K pol gene andprotein.

Example 15 Further Identification of Prostate-Specific Antigens byMicroarray Analysis

This Example describes the isolation of additional prostate-specificpolypeptides from a prostate tumor cDNA library.

A human prostate tumor cDNA expression library as described above wasscreened using microarray analysis to identify clones that display atleast a three fold over-expression in prostate tumor and/or normalprostate tissue, as compared to non-prostate normal tissues (notincluding testis). 142 clones were identified and sequenced. Certain ofthese clones are shown in SEQ ID NO: 454-467. Of these sequences, SEQ IDNO: 459-460 represent novel genes. The others (SEQ ID NO: 454-458 and461-467) correspond to known sequences. Comparison of the determinedcDNA sequence of SEQ ID NO: 461 with sequences in the Genbank databaseusing the BLAST program revealed homology to the previously identifiedtransmembrane protease serine 2 (TMPRSS2). The full-length cDNA sequencefor this clone is provided in SEQ ID NO: 894, with the correspondingamino acid sequence being provided in SEQ ID NO: 895. The cDNA sequenceencoding the first 209 amino acids of TMPRSS2 is provided in SEQ ID NO:896, with the first 209 amino acids being provided in SEQ ID NO: 897.

The sequence of SEQ ID NO: 462 (referred to as P835P) was found tocorrespond to the previously identified clone FLJ13518 (AccessionAK023643; SEQ ID NO: 917), which had no associated open reading frame(ORF). This clone was used to search the Geneseq DNA database andmatched a clone previously identified as a G protein-coupled receptorprotein (DNA Geneseq Accession A09351; amino acid Geneseq AccessionY92365), that is characterized by the presence of seven transmembranedomains. The sequences of fragments between these domains are providedin SEQ ID NO: 921-928, with SEQ ID NO: 921, 923, 925 and 927representing extracellular domains and SEQ ID NO: 922, 924, 926 and 928representing intracellular domains. SEQ ID NO: 921-928 represent aminoacids 1-28, 53-61, 83-103, 124-143, 165-201, 226-238, 263-272 and297-381, respectively, of P835P. The full-length cDNA sequence for P835Pis provided in SEQ ID NO: 916. The cDNA sequence of the open readingframe for P835P, including stop codon, is provided in SEQ ID NO: 918,with the open reading frame without stop codon being provided in SEQ IDNO: 919 and the corresponding amino acid sequence being provided in SEQID NO: 920.

Example 16 Further Characterization of Prostate-Specific Antigen P710P

This Example describes the full length cloning of P710P.

The prostate cDNA library described above was screened with the P710Pfragment described above. One million colonies were plated onLB/Ampicillin plates. Nylon membrane filters were used to lift thesecolonies, and the cDNAs picked up by these filters were then denaturedand cross-linked to the filters by UV light. The P710P fragment wasradiolabeled and used to hybridize with the filters. Positive cDNAclones were selected and their cDNAs recovered and sequenced by anautomatic Perkin Elmer/Applied Biosystems Division Sequencer. Foursequences were obtained, and are presented in SEQ ID NO: 468-471. Thesesequences appear to represent different splice variants of the P710Pgene. Subsequent comparison of the cDNA sequences of P710P with those inGenbank releaved homology to the DD3 gene (Genbank accession numbersAF103907 & AF103908). The cDNA sequence of DD3 is provided in SEQ ID NO:690.

Example 17 Protein Expression of Prostate-Specific Antigens

This example describes the expression and purification ofprostate-specific antigens in E. Coli baculovirus and mammalian cells.

a) Expression of P501S in E. coli

Expression of the full-length form of P501S was attempted by firstcloning P501S without the leader sequence (amino acids 36-553 of SEQ IDNO: 113) downstream of the first 30 amino acids of the M. tuberculosisantigen Ra12 (SEQ ID NO: 484) in pET17b. Specifically, P501S DNA wasused to perform PCR using the primers AW025 (SEQ ID NO: 485) and AW003(SEQ ID NO: 486). AW025 is a sense cloning primer that contains aHindIII site. AW003 is an antisense cloning primer that contains anEcoRI site. DNA amplification was performed using 5 μl 10×Pfu buffer,1120 mM dNTPs, 1 μl each of the PCR primers at 10 μM concentration, 40μl water, 1 μl Pfu DNA polymerase (Stratagene, La Jolla, Calif.) and 1μl DNA at 100 ng/μl. Denaturation at 95° C. was performed for 30 sec,followed by 10 cycles of 95° C. for 30 sec, 60° C. for 1 min and by 72°C. for 3 min. 20 cycles of 95° C. for 30 sec, 65° C. for 1 min and by72° C. for 3 min, and lastly by 1 cycle of 72° C. for 10 min. The PCRproduct was cloned to Ra12m/pET17b using HindIII and EcoRI. The sequenceof the resulting fusion construct (referred to as Ra12-P501S-F) wasconfirmed by DNA sequencing.

The fusion construct was transformed into BL21 (DE3)pLysE, pLysS andCodonPlus E. coli (Stratagene) and grown overnight in LB broth withkanamycin. The resulting culture was induced with IPTG. Protein wastransferred to PVDF membrane and blocked with 5% non-fat milk (inPBS-Tween buffer), washed three times and incubated with mouse anti-Histag antibody (Clontech) for 1 hour. The membrane was washed 3 times andprobed with HRP-Protein A (Zymed) for 30 min. Finally, the membrane waswashed 3 times and developed with ECL (Amersham). No expression wasdetected by Western blot. Similarly, no expression was detected byWestern blot when the Ra12-P501S-F fusion was used for expression inBL21 CodonPlus by CE6 phage (Invitrogen).

An N-terminal fragment of P501S (amino acids 36-325 of SEQ ID NO: 113)was cloned down-stream of the first 30 amino acids of the M.tuberculosis antigen Ra12 in pET17b as follows. P501S DNA was used toperform PCR using the primers AW025 (SEQ ID NO: 485) and AW027 (SEQ IDNO: 487). AW027 is an antisense cloning primer that contains an EcoRIsite and a stop codon. DNA amplification was performed essentially asdescribed above. The resulting PCR product was cloned to Ra12 in pET17bat the HindIII and EcoRI sites. The fusion construct (referred to asRa12-P501S-N) was confirmed by DNA sequencing.

The Ra12-P501S-N fusion construct was used for expression in BL21(DE3)pLysE, pLysS and CodonPlus, essentially as described above. UsingWestern blot analysis, protein bands were observed at the expectedmolecular weight of 36 kDa. Some high molecular weight bands were alsoobserved, probably due to aggregation of the recombinant protein. Noexpression was detected by Western blot when the Ra12-P501S-F fusion wasused for expression in BL21CodonPlus by CE6 phage.

A fusion construct comprising a C-terminal portion of P501S (amino acids257-553 of SEQ ID NO: 113) located down-stream of the first 30 aminoacids of the M. tuberculosis antigen Ra12 (SEQ ID NO: 484) was preparedas follows. P501S DNA was used to perform PCR using the primers AW026(SEQ ID NO: 488) and AW003 (SEQ ID NO: 486). AW026 is a sense cloningprimer that contains a HindIII site. DNA amplification was performedessentially as described above. The resulting PCR product was cloned toRa12 in pET17b at the HindIII and EcoRI sites. The sequence for thefusion construct (referred to as Ra12-P501S-C) was confirmed.

The Ra12-P501S-C fusion construct was used for expression in BL21(DE3)pLysE, pLysS and CodonPlus, as described above. A small amount ofprotein was detected by Western blot, with some molecular weightaggregates also being observed. Expression was also detected by Westernblot when the Ra12-P501S-C fusion was used for expression in BL21CodonPlus induced by CE6 phage.

A fusion construct comprising a fragment of P501S (amino acids 36-298 ofSEQ ID NO: 113) located down-stream of the M. tuberculosis antigen Ra12(SEQ ID NO: 848) was prepared as follows. P501S DNA was used to performPCR using the primers AW042 (SEQ ID NO: 849) and AW053 (SEQ ID NO: 850).AW042 is a sense cloning primer that contains a EcoRI site. AW053 is anantisense primer with stop and Xho I sites. DNA amplification wasperformed essentially as described above. The resulting PCR product wascloned to Ra12 in pET17b at the EcoRI and Xho I sites. The resultingfusion construct (referred to as Ra12-P501S-E2) was expressed in B834(DE3) pLys S E. coli host cells in TB media for 2 h at room temperature.Expressed protein was purified by washing the inclusion bodies andrunning on a Ni-NTA column. The purified protein stayed soluble inbuffer containing 20 mM Tris-HCl (pH 8), 100 mM NaCl, 10 mM β-Me and 5%glycerol. The determined cDNA and amino acid sequences for the expressedfusion protein are provided in SEQ ID NO: 851 and 852, respectfully.

b) Expression of P501S in Baculovirus

The Bac-to-Bac baculovirus expression system (BRL Life Technologies,Inc.) was used to express P501S protein in insect cells. Full-lengthP501S (SEQ ID NO: 113) was amplified by PCR and cloned into the XbaIsite of the donor plasmid pFastBacl. The recombinant bacmid andbaculovirus were prepared according to the manufacturer's instructions.The recombinant baculovirus was amplified in Sf9 cells and the hightiter viral stocks were utilized to infect High Five cells (Invitrogen)to make the recombinant protein. The identity of the full-length proteinwas confirmed by N-terminal sequencing of the recombinant protein and byWestern blot analysis (FIG. 7). Specifically, 0.6 million High Fivecells in 6-well plates were infected with either the unrelated controlvirus BV/ECD_PD (lane 2), with recombinant baculovirus for P501S atdifferent amounts or MOIs (lanes 4-8), or were uninfected (lane 3). Celllysates were run on SDS-PAGE under reducing conditions and analyzed byWestern blot with the anti-P501S monoclonal antibody P501S-10E3-G4D3(prepared as described below). Lane 1 is the biotinylated proteinmolecular weight marker (BioLabs).

The localization of recombinant P501S in the insect cells wasinvestigated as follows. The insect cells overexpressing P501S werefractionated into fractions of nucleus, mitochondria, membrane andcytosol. Equal amounts of protein from each fraction were analyzed byWestern blot with a monoclonal antibody against P501S. Due to the schemeof fractionation, both nucleus and mitochondria fractions contain someplasma membrane components. However, the membrane fraction is basicallyfree from mitochondria and nucleus. P501S was found to be present in allfractions that contain the membrane component, suggesting that P501S maybe associated with plasma membrane of the insect cells expressing therecombinant protein.

c) Expression of P501S in Mammalian Cells

Full-length P501S (553 amino acids; SEQ ID NO: 113) was cloned intovarious mammalian expression vectors, including pCEP4 (Invitrogen),pVR1012 (Vical, San Diego, Calif.) and a modified form of the retroviralvector pBMN, referred to as pBIB. Transfection of P501S/pCEP4 andP501S/pVR1012 into HEK293 fibroblasts was carried out using the Fugenetransfection reagent (Boehringer Mannheim). Briefly, 2 ul of Fugenereagent was diluted into 100 ul of serum-free media and incubated atroom temperature for 5-10 min. This mixture was added to 1 ug of P501Splasmid DNA, mixed briefly and incubated for 30 minutes at roomtemperature. The Fugene/DNA mixture was added to cells and incubated for24-48 hours. Expression of recombinant P501S in transfected HEK293fibroblasts was detected by means of Western blot employing a monoclonalantibody to P501S.

Transfection of p501S/pCEP4 into CHO-K cells (American Type CultureCollection, Rockville, Md.) was carried out using GenePortertransfection reagent (Gene Therapy Systems, San Diego, Calif.). Briefly,15 μl of GenePorter was diluted in 500 μl of serum-free media andincubated at room temperature for 10 min. The GenePorter/media mixturewas added to 2 μg of plasmid DNA that was diluted in 500 μl ofserum-free media, mixed briefly and incubated for 30 min at roomtemperature. CHO-K cells were rinsed in PBS to remove serum proteins,and the GenePorter/DNA mix was added and incubated for 5 hours. Thetransfected cells were then fed an equal volume of 2× media andincubated for 24-48 hours.

FACS analysis of P501S transiently infected CHO-K cells, demonstratedsurface expression of P501S. Expression was detected using rabbitpolyclonal antisera raised against a P501S peptide, as described below.Flow cytometric analysis was performed using a FaCScan (BectonDickinson), and the data were analyzed using the Cell Quest program.

d) Expression of P703P in Baculovirus

The cDNA for full-length P703P-DE5 (SEQ ID NO: 326), together withseveral flanking restriction sites, was obtained by digesting theplasmid pCDNA703 with restriction endonucleases Xba I and Hind III. Theresulting restriction fragment (approx. 800 base pairs) was ligated intothe transfer plasmid pFastBacl which was digested with the samerestriction enzymes. The sequence of the insert was confirmed by DNAsequencing. The recombinant transfer plasmid pFBP703 was used to makerecombinant bacmid DNA and baculovirus using the Bac-To-Bac Baculovirusexpression system (BRL Life Technologies). High Five cells were infectedwith the recombinant virus BVP703, as described above, to obtainrecombinant P703P protein.

e) Expression of P788P in E. Coli

A truncated, N-terminal portion, of P788P (residues 1-644 of SEQ ID NO:777; referred to as P788P-N) fused with a C-terminal 6×His Tag wasexpressed in E. coli as follows. P788P cDNA was amplified using theprimers AW080 and AW081 (SEQ ID NO: 815 and 816). AW080 is a sensecloning primer with an NdeI site. AW081 is an antisense cloning primerwith a XhoI site. The PCR-amplified P788P, as well as the vector pCRX1,were digested with NdeI and XhoI. Vector and insert were ligated andtransformed into NovaBlue cells. Colonies were randomly screened forinsert and then sequenced. P788P-N clone #6 was confirmed to beidentical to the designed construct. The expression construct P788P-N#6/pCRX1 was transformed into E. coli BL21 CodonPlus-RIL competentcells. After induction, most of the cells grew well, achieving OD600 ofgreater than 2.0 after 3 hr. Coomassie stained SDS-PAGE showed anover-expressed band at about 75 kD. Western blot analysis using a6×HisTag antibody confirmed the band was P788P-N. The determined cDNAsequence for P788P-N is provided in SEQ ID NO: 817, with thecorresponding amino acid sequence being provided in SEQ ID NO: 818.

f) Expression of P510S in E. coli

The P510S protein has 9 potential transmembrane domains and is predictedto be located at the plasma membrane. The C-terminal protein of thisprotein, as well as the predicted third extracellular domain of P510Swere expressed in E. coli as follows.

The expression construct referred to as Ra12-P501S-C was designed tohave a 6 HisTag at the N-terminal enc, followed by the M. tuberculosisantigen Ra12 (SEQ ID NO: 819) and then the C-terminal portion of P510S(amino residues 1176-1261 of SEQ ID NO: 538). Full-length P510S was usedto amplify the P510S-C fragment by PCR using the primers AW056 and AW057(SEQ ID NO: 820 and 821, respectively). AW056 is a sense cloning primerwith an EcoRI site. AW057 is an antisense primer with stop and XhoIsites. The amplified P501S fragment and Ra12/pCRX1 were digested withEcoRI and XhoI and then purified. The insert and vector were ligatedtogether and transformed into NovaBlue. Colonies were randomly screenedfor insert and sequences. For protein expression, the expressionconstruct was transformed into E. coli BL21 (DE3) CodonPlus-RILcompetent cells. A mini-induction screen was performed to optimize theexpression conditions. After induction the cells grew well, achieving OD600 nm greater than 2.0 after 3 hours. Coomassie stain SDS-PAGE showed ahighly over-expressed band at approx. 30 kD. Though this is higher thanthe expected molecular weight, western blot analysis was positive,showing this band to be the His tag-containing protein. The optimizedculture conditions are as follows. Dilute overnight culture/daytimeculture (LB+kanamycin+chloramphenicol) into 2xYT (with kanamycin andchloramphenicol) at a ratio of 25 ml culture to 1 liter 2xYT. Allow togrow at 37° C. until OD600=0.6. Take an aliquot out as TO sample. Add 1mM IPTG and allow to grow at 30° C. for 3 hours. Take out a T3 sample,spin down cells and store at −80° C. The determined cDNA and amino acidsequences for the Ra12-P510S-C construct are provided in SEQ ID NO: 822and 825, respectively.

The expression construct P510S-C was designed to have a 5′ added startcodon and a glycine (GGA) codon and then the P510S C terminal fragmentfollowed by the in frame 6× histidine tag and stop codon from the pET28bvector. The cloning strategy is similar to that used for Ra12-P510S-C,except that the PCR primers employed were those shown in SEQ ID NO: 828and 829, respectively and the NcoI/XhoI cut in pET28b was used. Theprimer of SEQ ID NO: 828 created a 5′ NcoI site and added a start codon.The antisense primer of SEQ ID NO: 829 creates a XhoI site on P510S Cterminal fragment. Clones were confirmed by sequencing. For proteinexpression, the expression construct was transformed into E. coli BL21(DE3) CodonPlus-RIL competent cells. An OD600 of greater than 2.0 wasobtained 30 hours after induction. Coomassie stained SDS-PAGE showed anover-expressed band at about 11 kD. Western blot analysis confirmed thatthe band was P510S-C, as did N-terminal protein sequencing. Theoptimized culture conditions are as follows: dilute overnightculture/daytime culture (LB+kanamycin+chloramphenicol) into 2x YT(+kanamycin and chloramphenicol) at a ratio of 25 mL culture to 1 liter2x YT, and allow to grow at 37° C. until an OD 600 of about 0.5 isreached. Take out an aliquot as TO sample. Add 1 mM IPTG and allow togrow at 30° C. for 3 hours. Spin down the cells and store at −80° C.until purification. The determined cDNA and amino acid sequences for theP510S-C construct are shown in SEQ ID NO: 823 and 826, respectively.

The predicted third extracellular domain of P510S(P510S-E3; residues328-676 of SEQ ID NO: 538) was expressed in E. coli as follows. TheP510S fragment was amplified by PCR using the primers shown in SEQ IDNO: 830 and 831. The primer of SEQ ID NO: 830 is a sense primer with anNdeI site for use in ligating into pPDM. The primer of SEQ ID NO: 831 isan antisense primer with an added XhoI site for use in ligating intopPDM. The resulting fragment was cloned to pPDM at the NdeI and XhoIsites. Clones were confirmed by sequencing. For protein expression, theclone ws transformed into E. coli BL21 (DE3) CodonPlus-RIL competentcells. After induction, an OD600 of greater than 2.0 was achieved after3 hours. Coomassie stained SDS-PAGE showed an over-expressed band atabout 39 kD, and N-terminal sequencing confirmed the N-terminal to bethat of P510S-E3. Optimized culture conditions are as follows: diluteovernight culture/daytime culture (LB+kanamycin+chloramphenicol) into 2xYT (kanamycin and chloramphenicol) at a ratio of 25 ml culture to 1liter 2x YT. Allow to grow at 37° C. until OD 600 equals 0.6. Take outan aliquot as TO sample. Add 1 mM IPTG and allow to grow at 30° C. for 3hours. Take out a T3 sample, spin down the cells and store at −80° C.until purification. The determined cDNA and amino acid sequences for theP501S-E3 construct are provided in SEQ ID NO: 824 and 827, respectively.

g) Expression of P775S in E. coli

The antigen P775P contains multiple open reading frames (ORF). The thirdORF, encoding the protein of SEQ ID NO: 483, has the best emotif score.An expression fusion construct containing the M. tuberculosis antigenRa12 (SEQ ID NO: 819) and P775P-ORF3 with an N-terminal 6×HisTag wasprepared as follows. P775P-ORF3 was amplified using the sense PCRprimers of SEQ ID NO: 832 and the anti-sense PCR primer of SEQ ID NO:833. The PCR amplified fragment of P775P and Ra12/pCRX1 were digestedwith the restriction enzymes EcoRI and XhoI. Vector and insert wereligated and then transformed into NovaBlue cells. Colonies were randomlyscreened for insert and then sequenced. A clone having the desiredsequence was transformed into E. coli BL21 (DE3) CodonPlus-RIL competentcells. Two hours after induction, the cell density peaked at OD600 ofapproximately 1.8. Coomassie stained SDS-PAGE showed an over-expressedband at about 31 kD. Western blot using 6×HisTag antibody confirmed thatthe band was Ra12-P775P-ORF3. The determined cDNA and amino acidsequences for the fusion construct are provided in SEQ ID NO: 834 and835, respectively.

h) Expression of a P703P His Tag Fusion Protein in E. coli

The cDNA for the coding region of P703P was prepared by PCR using theprimers of SEQ ID NO: 836 and 837. The PCR product was digested withEcoRI restriction enzyme, gel purified and cloned into a modified pET28vector with a His tag in frame, which had been digested with Eco72I andEcoRI restriction enzymes. The correct construct was confirmed by DNAsequence analysis and then transformed into E. coli BL21 (DE3) pLys Sexpression host cells. The determined amino acid and cDNA sequences forthe expressed recombinant P703P are provided in SEQ ID NO: 838 and 839,respectively.

i) Expression of a P705P His Tag Fusion Protein in E. coli

The cDNA for the coding region of P705P was prepared by PCR using theprimers of SEQ ID NO: 840 and 841. The PCR product was digested withEcoRI restriction enzyme, gel purified and cloned into a modified pET28vector with a His tag in frame, which had been digested with Eco721I andEcoRI restriction enzymes. The correct construct was confirmed by DNAsequence analysis and then transformed into E. coli BL21 (DE3) pLys Sand BL21 (DE3) CodonPlus expression host cells. The determined aminoacid and cDNA sequences for the expressed recombinant P705P are providedin SEQ ID NO: 842 and 843, respectively.

j) Expression of a P711P His Tag Fusion Protein in E. coli

The cDNA for the coding region of P711P was prepared by PCR using theprimers of SEQ ID NO: 844 and 845. The PCR product was digested withEcoRI restriction enzyme, gel purified and cloned into a modified pET28vector with a His tag in frame, which had been digested with Eco721I andEcoRI restriction enzymes. The correct construct was confirmed by DNAsequence analysis and then transformed into E. coli BL21 (DE3) pLys Sand BL21 (DE3) CodonPlus expression host cells. The determined aminoacid and cDNA sequences for the expressed recombinant P711P are providedin SEQ ID NO: 846 and 847, respectively.

k) Expression of P767P in E. coli

The full-length coding region of P767P (amino acids 2-374 of SEQ ID NO:590) was amplified by PCR using the primers PDM-468 and PDM-469 (SEQ IDNO: 935 and 936, respectively). DNA amplification was performed using 10μl 10×Pfu buffer, 1 μl 10 mM dNTPs, 2 μl each of the PCR primers at 10μM concentration, 83 μl water, 1.5 μl Pfu DNA polymerase (Stratagene, LaJolla, Calif.) and 1 μl DNA at 100 ng/μl. Denaturation at 96° C. wasperformed for 2 min, followed by 40 cycles of 96° C. for 20 sec, 66° C.for 15 sec and by 72° C. for 2 min., and lastly by 1 cycle of 72° C. for4 min. The PCR product was digested with XhoI and cloned into a modifiedpET28 vector with a histidine tag in frame on the 5′ end that wasdigested with Eco721I and XhoI. The construct was confirmed to becorrect through sequence analysis and transformed into E. coli BL21pLysS and BL21 CodonPlus RP cells. The cDNA coding region for therecombinant B767P protein is provided in SEQ ID NO: 938, with thecorresponding amino acid sequence being provided in SEQ ID NO: 941. Thefull-length P767P did not express at high enough levels for detection orpurification.

A truncated coding region of P767P (referred to as B767P-B; amino acids47-374 of SEQ ID NO: 590) was amplified by PCR using the primers PDM-573and PDM-469 (SEQ ID NO: 937 and 936, respectively) and the PCRconditions described above for full-length P767P. The PCR product wasdigested with XhoI and cloned into the modified pET28 vector that wasdigested with Eco721I and XhoI. The construct was confirmed to becorrect through sequence analysis and transformed into E. coli BL21pLysS and BL21 CodonPlus RP cells. The protein was found to be expressedin the inclusion body pellet. The coding region for the expressedB767P-B protein is provided in SEQ ID NO: 939, with the correspondingamino acid sequence being provided in SEQ ID NO: 940.

l) Expression of P767P in Mammalian Cells

For recombinant expression in mammalian cells, the full length P767PcDNA (SEQ ID NO: 587) was subcloned into the mammalian expression vectorpCEP4 (Invitrogen) that was modified to contain a FLAG epitope tag. Thisconstruct was transfected into HEK293 cells (American Type CultureCollection) using Fugene 6 reagent (Roche). Briefly, the HEK cells wereplated at a density of 100,000 cells/ml in DMEM (Gibco) containing 10%FBS (Hyclone) and grown overnight. The following day, 2 ul of Fugene 6was added to 100 ul of DMEM containing no FBS and incubated for 15minutes at room temperature. The Fugene 6/DMEM mixture was then added to1 ug of P767P/pCEP4 plasmid DNA and incubated for 15 minutes at roomtemperature. The Fugene/DNA mix was then added to the HEK293 cells andincubated for 48-72 hrs at 37° C. with 7% CO₂. Cells were rinsed withPBS then collected and pelleted by centrifugation.

P767P expression was detect in transfected HEK293 whole cell lysates byWestern blot analysis using an anti-epitope tag antibody. Specifically,whole cell lysates were generated by incubating the cells in Triton-X100containing lysis buffer for 30 minutes on ice. Lysates were then clearedby centrifugation at 5,000 rpm for 5 minutes at 4° C. Samples werediluted with SDS-PAGE loading buffer containing beta-mercaptoethanol,then boiled for 10 minutes prior to loading the SDS-PAGE gel. Proteinwas transferred to nitrocellulose and probed using an anti-FLAG mousemonoclonal antibody (Sigma) at a dilution of 1 ug/ml. The blot wasrevealed with a donkey anti-mouse Ig coupled to HRP followed byincubation in ECL substrate.

P767P was found to be expressed on the surface of transfected HEK293cells by flow cytometry using rabbit anti-P767P sera as follows. Cellswere collected and washed with ice cold staining buffer (PBS+1%BSA+Azide). Next, the cells were incubated for 30 minutes on ice withundiluted anti-P767P rabbit polyclonal sera. The cells were washed 3times with staining buffer and then incubated with a 1:100 dilution of agoat anti-rabbit Ig(H+L)-FITC reagent (Southern Biotechnology) for 30minutes on ice. Following 3 washes, the cells were resuspended instaining buffer containing Propidium Iodide (PI), a vital stain thatallows for identification of permeable cells, and analyzed by FACS.

m) Expression of P788P in Mammalian Cells

For recombinant expression in mammalian cells, the full length P788PcDNA was subcloned into the mammalian expression vector pcDNA3.1(Invitrogen). This construct was transfected into HEK293 cells (AmericanType Culture Collection) using Fugene 6 reagent (Roche). Briefly, theHEK cells were plated at a density of 100,000 cells/ml in DMEM (Gibco)containing 10% FBS (Hyclone) and grown overnight. The following day, 2ul of Fugene6 was added to 100 ul of DMEM containing no FBS andincubated for 15 minutes at room temperature. The Fugene6/DMEM mixturewas then added to 1 ug of P788P/pcDNA3.1 plasmid DNA and incubated for15 minutes at room temperature. The Fugene/DNA mix was then added to theHEK293 cells and incubated for 48-72 hrs at 37° C. with 7% CO₂. Cellswere rinsed with PBS then collected and pelleted by centrifugation.

P788P expression was detected in transfected HEK293 whole cell lysatesby Western blot analysis using rabbit anti-P788P sera as probe.Specifically, whole cell lysates were generated by incubating the cellsin Triton-X100 containing lysis buffer for 30 minutes on ice. Lysateswere then cleared by centrifugation at 5,000 rpm for 5 minutes at 4° C.Samples were diluted with SDS-PAGE loading buffer containingbeta-mercaptoethanol, then boiled for 10 minutes prior to loading theSDS-PAGE gel. Protein was transferred to nitrocellulose and probed usingpurified anti-P788P rabbit polyclonal sera at a concentration of 1ug/ml. The blot was revealed with a donkey anti-rabbit Ig coupled to HRP(Jackson ImmunoResearch) followed by incubation in ECL substrate.

P788P expression was detected on the surface of transfected HEK293 cellsby flow cytometry using rabbit anti-P788P sera. For FACS analysis, cellswere collected and washed with ice cold staining buffer (PBS+1%BSA+Azide). Next, the cells were incubated for 30 minutes on ice withanti-P788P polyclonal sera (10 ug/ml final concentration). The cellswere washed 3 times with staining buffer and then incubated with a 1:100dilution of a goat anti-rabbit Ig(H+L)-FITC reagent (SouthernBiotechnology) for 30 minutes on ice. Following 3 washes, the cells wereresuspended in staining buffer containing Propidium Iodide (PI), a vitalstain that allows for identification of permeable cells, and analyzed byflow cytometry.

Example 18 Preparation and Characterization of Antibodies AgainstProstate-Specific Polypeptides

a) Preparation and Characterization of Polyclonal Antibodies againstP703P, P504S and P509S

Polyclonal antibodies against P703P, P504S, P509S, P501S, P510S-E3,P510S-C, P705P, full-length P703P (referred to as P703 PFL), P711P,P767P, P788P-N, and peptides of P790P and P775P were prepared asfollows. The amino acid sequences of the P790P peptides (referred to asP790P89, P790P120, P790P163, P790P180, P790P222, P790P261, P790P287 andP790P307, respectively) are provided in SEQ ID NO: 949-956, with theamino acid sequences of the P775P peptides being provided in SEQ ID NO:957-966.

Each prostate tumor antigen expressed in an E. coli recombinantexpression system was grown overnight in LB broth with the appropriateantibiotics at 37° C. in a shaking incubator. The next morning, 10 ml ofthe overnight culture was added to 500 ml to 2x YT plus appropriateantibiotics in a 2 L-baffled Erlenmeyer flask. When the Optical Density(at 560 nm) of the culture reached 0.4-0.6, the cells were induced withIPTG (1 mM). Four hours after induction with IPTG, the cells wereharvested by centrifugation. The cells were then washed with phosphatebuffered saline and centrifuged again. The supernatant was discarded andthe cells were either frozen for future use or immediately processed.Twenty ml of lysis buffer was added to the cell pellets and vortexed. Tobreak open the E. coli cells, this mixture was then run through theFrench Press at a pressure of 16,000 psi. The cells were thencentrifuged again and the supernatant and pellet were checked bySDS-PAGE for the partitioning of the recombinant protein. For proteinsthat localized to the cell pellet, the pellet was resuspended in 10 mMTris pH 8.0, 1% CHAPS and the inclusion body pellet was washed andcentrifuged again. This procedure was repeated twice more. The washedinclusion body pellet was solubilized with either 8 M urea or 6 Mguanidine HCl containing 10 mM Tris pH 8.0 plus 10 mM imidazole. Thesolubilized protein was added to 5 ml of nickel-chelate resin (Qiagen)and incubated for 45 min to 1 hour at room temperature with continuousagitation. After incubation, the resin and protein mixture were pouredthrough a disposable column and the flow through was collected. Thecolumn was then washed with 10-20 column volumes of the solubilizationbuffer. The antigen was then eluted from the column using 8M urea, 10 mMTris pH 8.0 and 300 mM imidazole and collected in 3 ml fractions. ASDS-PAGE gel was run to determine which fractions to pool for furtherpurification.

As a final purification step, a strong anion exchange resin such asHiPrepQ (Biorad) was equilibrated with the appropriate buffer and thepooled fractions from above were loaded onto the column. Each antigenwas eluted off the column with a increasing salt gradient. Fractionswere collected as the column was run and another SDS-PAGE gel was run todetermine which fractions from the column to pool. The pooled fractionswere dialyzed against 10 mM Tris pH 8.0. The proteins were then vialedafter filtration through a 0.22 micron filter and the antigens werefrozen until needed for immunization.

The P790P and P775P peptides were synthesized, conjugated to KLH andfrozen until needed for immunization.

Four hundred micrograms of each prostate antigen or peptide was combinedwith 100 micrograms of muramyldipeptide (MDP) and injected into arabbit. Every four weeks rabbits were boosted with 100 micrograms ofantigen/peptide mixed with an equal volume of Incomplete Freund'sAdjuvant (IFA). Seven days following each boost, the animal was bled.Sera were generated by incubating the blood at 4° C. for 12-24 hoursfollowed by centrifugation.

Ninety-six well plates were coated with antigen by incubating with 50microliters (typically 1 microgram/microliter) of recombinant protein at4° C. for 20 hours. 250 microliters of BSA blocking buffer was added tothe wells and incubated at room temperature for 2 hours. Plates werewashed 6 times with PBS/0.1% Tween. Rabbit sera was diluted in PBS/0.1%Tween/0.1% BSA. Fifty microliters of diluted sera was added to each welland incubated at room temperature for 30 min. Plates were washed asdescribed above before 50 microliters of goat anti-rabbit horse radishperoxidase (HRP) at a 1:10000 dilution was added and incubated at roomtemperature for 30 min. Plates were again washed as described above and100 microliters of TMB microwell peroxidase substrate was added to eachwell. Following a 15 min incubation in the dark at room temperature, thecolorimetric reaction was stopped with 100 microliters of 1N H₂SO₄ andread immediately at 450 nm. All polyclonal antibodies showedimmunoreactivity to the appropriate antigen or peptide.

In order to determine which tissues express P509S, immunohistochemistry(IHC) analysis was performed on a diverse range of tissue sections.Tissue samples were fixed in formalin solution for 12-24 hrs andembedded in paraffin before being sliced into 8 micron sections. Steamheat induced epitope retrieval (SHIER) in 0.1 M sodium citrate buffer(pH 6.0) was used for optimal staining conditions. Sections wereincubated with 10% serum/PBS for 5 minutes. Primary antibody was addedto each section for 25 minutes at indicated concentrations followed by25 minute incubation with either anti-rabbit or anti-mouse biotinylatedantibody. Endogenous peroxidase activity was blocked by three 1.5 minuteincubations with hydrogen peroxidase. The avidin biotin complex/horseradish peroxidase (ABC/HRP) system was used along with DAB chromogen tovisualize antigen expression. Slides were counterstained withhematoxylin to visualize cell nuclei. P509S expression was detected inprostate cancer, BPH, colon, and kidney but not in heart, lung andliver.

b) Preparation and Characterization of Antibodies Against P501S

A murine monoclonal antibody directed against the carboxy-terminus ofthe prostate-specific antigen P501S was prepared as follows.

A truncated fragment of P501S (amino acids 355-526 of SEQ ID NO: 113)was generated and cloned into the pET28b vector (Novagen) and expressedin E. coli as a thioredoxin fusion protein with a histidine tag. Thetrx-P501S fusion protein was purified by nickel chromatography, digestedwith thrombin to remove the trx fragment and further purified by an acidprecipitation procedure followed by reverse phase HPLC.

Mice were immunized with truncated P501S protein. Serum bleeds from micethat potentially contained anti-P501S polyclonal sera were tested forP501S-specific reactivity using ELISA assays with purified P501S andtrx-P501S proteins. Serum bleeds that appeared to react specificallywith P501S were then screened for P501S reactivity by Western analysis.Mice that contained a P501S-specific antibody component were sacrificedand spleen cells were used to generate anti-P501S antibody producinghybridomas using standard techniques. Hybridoma supernatants were testedfor P501S-specific reactivity initially by ELISA, and subsequently byFACS analysis of reactivity with P501S transduced cells. Based on theseresults, a monoclonal hybridoma referred to as 10E3 was chosen forfurther subcloning. A number of subclones were generated, tested forspecific reactivity to P501S using ELISA and typed for IgG isotype. Theresults of this analysis are shown below in Table V. Of the 16 subclonestested, the monoclonal antibody 10E3-G4-D3 was selected for furtherstudy.

TABLE VI ISOTYPE ANALYSIS OF MURINE ANTI-P501S MONOCLONAL ANTIBODIESEstimated [Ig] in supernatant Hybridoma clone Isotype (μg/ml) 4D11 IgG114.6 1G1 IgG1 0.6 4F6 IgG1 72 4H5 IgG1 13.8 4H5-E12 IgG1 10.7 4H5-EH2IgG1 9.2 4H5-H2-A10 IgG1 10 4H5-H2-A3 IgG1 12.8 4H5-H2-A10-G6 IgG1 13.64H5-H2-B11 IgG1 12.3 10E3 IgG2a 3.4 10E3-D4 IgG2a 3.8 10E3-D4-G3 IgG2a9.5 10E3-D4-G6 IgG2a 10.4 10E3-E7 IgG2a 6.5 8H12 IgG2a 0.6

The specificity of 10E3-G4-D3 for P501S was examined by FACS analysis.Specifically, cells were fixed (2% formaldehyde, 10 minutes),permeabilized (0.1% saponin, 10 minutes) and stained with 10E3-G4-D3 at0.5-1 μg/ml, followed by incubation with a secondary, FITC-conjugatedgoat anti-mouse Ig antibody (Pharmingen, San Diego, Calif.). Cells werethen analyzed for FITC fluorescence using an Excalibur fluorescenceactivated cell sorter. For FACS analysis of transduced cells, B-LCL wereretrovirally transduced with P501S. For analysis of infected cells,B-LCL were infected with a vaccinia vector that expresses P501S. Todemonstrate specificity in these assays, B-LCL transduced with adifferent antigen (P703P) and uninfected B-LCL vectors were utilized.10E3-G4-D3 was shown to bind with P501S-transduced B-LCL and also withP501S-infected B-LCL, but not with either uninfected cells orP703P-transduced cells.

To determine whether the epitope recognized by 10E3-G4-D3 was found onthe surface or in an intracellular compartment of cells, B-LCL weretransduced with P501S or HLA-B8 as a control antigen and either fixedand permeabilized as described above or directly stained with 10E3-G4-D3and analyzed as above. Specific recognition of P501S by 10E3-G4-D3 wasfound to require permeabilization, suggesting that the epitoperecognized by this antibody is intracellular.

The reactivity of 10E3-G4-D3 with the three prostate tumor cell linesLncap, PC-3 and DU-145, which are known to express high, medium and verylow levels of P501S, respectively, was examined by permeabilizing thecells and treating them as described above. Higher reactivity of10E3-G4-D3 was seen with Lncap than with PC-3, which in turn showedhigher reactivity that DU-145. These results are in agreement with thereal time PCR and demonstrate that the antibody specifically recognizesP501S in these tumor cell lines and that the epitope recognized inprostate tumor cell lines is also intracellular.

Specificity of 10E3-G4-D3 for P501S was also demonstrated by Westernblot analysis. Lysates from the prostate tumor cell lines Lncap, DU-145and PC-3, from P501S-transiently transfected HEK293 cells, and fromnon-transfected HEK293 cells were generated. Western blot analysis ofthese lysates with 10E3-G4-D3 revealed a 46 kDa immunoreactive band inLncap, PC-3 and P501S-transfected HEK cells, but not in DU-145 cells ornon-transfected HEK293 cells. P501S mRNA expression is consistent withthese results since semi-quantitative PCR analysis revealed that P501SmRNA is expressed in Lncap, to a lesser but detectable level in PC-3 andnot at all in DU-145 cells. Bacterially expressed and purifiedrecombinant P501S (referred to as P501SStr2) was recognized by10E3-G4-D3 (24 kDa), as was full-length P501S that was transientlyexpressed in HEK293 cells using either the expression vector VR1012 orpCEP4. Although the predicted molecular weight of P501S is 60.5 kDa,both transfected and “native” P501S run at a slightly lower mobility dueto its hydrophobic nature.

Immunohistochemical analysis was performed on prostate tumor and a panelof normal tissue sections (prostate, adrenal, breast, cervix, colon,duodenum, gall bladder, ileum, kidney, ovary, pancreas, parotid gland,skeletal muscle, spleen and testis). Tissue samples were fixed informalin solution for 24 hours and embedded in paraffin before beingsliced into 10 micron sections. Tissue sections were permeabilized andincubated with 10E3-G4-D3 antibody for 1 hr. HRP-labeled anti-mousefollowed by incubation with DAB chromogen was used to visualize P501Simmunoreactivity. P501S was found to be highly expressed in both normalprostate and prostate tumor tissue but was not detected in any of theother tissues tested.

To identify the epitope recognized by 10E3-G4-D3, an epitope mappingapproach was pursued. A series of 13 overlapping 20-21 mers (5 aminoacid overlap; SEQ ID NO: 489-501) was synthesized that spanned thefragment of P501S used to generate 10E3-G4-D3. Flat bottom 96 wellmicrotiter plates were coated with either the peptides or the P501Sfragment used to immunize mice, at 1 microgram/ml for 2 hours at 37° C.Wells were then aspirated and blocked with phosphate buffered salinecontaining 1% (w/v) BSA for 2 hours at room temperature, andsubsequently washed in PBS containing 0.1% Tween 20 (PBST). Purifiedantibody 10E3-G4-D3 was added at 2 fold dilutions (1000 ng-16 ng) inPBST and incubated for 30 minutes at room temperature. This was followedby washing 6 times with PBST and subsequently incubating withHRP-conjugated donkey anti-mouse IgG (H+L) Affinipure F(ab′) fragment(Jackson Immunoresearch, West Grove, Pa.) at 1:20000 for 30 minutes.Plates were then washed and incubated for 15 minutes in tetramethylbenzidine. Reactions were stopped by the addition of 1N sulfuric acidand plates were read at 450 nm using an ELISA plate reader. As shown inFIG. 8, reactivity was seen with the peptide of SEQ ID NO: 496(corresponding to amino acids 439-459 of P501S) and with the P501Sfragment but not with the remaining peptides, demonstrating that theepitope recognized by 10E3-G4-D3 is localized to amino acids 439-459 ofSEQ ID NO: 113.

In order to further evaluate the tissue specificity of P501S,multi-array immunohistochemical analysis was performed on approximately4700 different human tissues encompassing all the major normal organs aswell as neoplasias derived from these tissues. Sixty-five of these humantissue samples were of prostate origin. Tissue sections 0.6 mm indiameter were formalin-fixed and paraffin embedded. Samples werepretreated with HIER using 10 mM citrate buffer pH 6.0 and boiling for10 min. Sections were stained with 10E3-G4-D3 and P501S immunoreactivitywas visualized with HRP. All the 65 prostate tissues samples (5 normal,55 untreated prostate tumors, 5 hormone refractory prostate tumors) werepositive, showing distinct perinuclear staining. All other tissuesexamined were negative for P501S expression.

c) Preparation and Characterization of Antibodies against P503S

A fragment of P503S (amino acids 113-241 of SEQ ID NO: 114) wasexpressed and purified from bacteria essentially as described above forP501S and used to immunize both rabbits and mice. Mouse monoclonalantibodies were isolated using standard hybridoma technology asdescribed above. Rabbit monoclonal antibodies were isolated usingSelected Lymphocyte Antibody Method (SLAM) technology at ImmgenicsPharmaceuticals (Vancouver, BC, Canada). Table VI, below, lists themonoclonal antibodies that were developed against P503S.

TABLE VII Antibody Species 20D4 Rabbit JA1 Rabbit 1A4 Mouse 1C3 Mouse1C9 Mouse 1D12 Mouse 2A11 Mouse 2H9 Mouse 4H7 Mouse 8A8 Mouse 8D10 Mouse9C12 Mouse 6D12 Mouse

The DNA sequences encoding the complementarity determining regions(CDRs) for the rabbit monoclonal antibodies 20D4 and JA1 were determinedand are provided in SEQ ID NO: 502 and 503, respectively.

In order to better define the epitope binding region of each of theantibodies, a series of overlapping peptides were generated that spanamino acids 109-213 of SEQ ID NO: 114. These peptides were used toepitope map the anti-P503S monoclonal antibodies by ELISA as follows.The recombinant fragment of P503S that was employed as the immunogen wasused as a positive control. Ninety-six well microtiter plates werecoated with either peptide or recombinant antigen at 20 ng/wellovernight at 4° C. Plates were aspirated and blocked with phosphatebuffered saline containing 1% (w/v) BSA for 2 hours at room temperaturethen washed in PBS containing 0.1% Tween 20 (PBST). Purified rabbitmonoclonal antibodies diluted in PBST were added to the wells andincubated for 30 min at room temperature. This was followed by washing 6times with PBST and incubation with Protein-A HRP conjugate at a 1:2000dilution for a further 30 min. Plates were washed six times in PBST andincubated with tetramethylbenzidine (TMB) substrate for a further 15min. The reaction was stopped by the addition of 1N sulfuric acid andplates were read at 450 nm using at ELISA plate reader. ELISA with themouse monoclonal antibodies was performed with supernatants from tissueculture run neat in the assay.

All of the antibodies bound to the recombinant P503S fragment, with theexception of the negative control SP2 supernatant. 20D4, JA1 and 1D12bound strictly to peptide #2101 (SEQ ID NO: 504), which corresponds toamino acids 151-169 of SEQ ID NO: 114. 1C3 bound to peptide #2102 (SEQID NO: 505), which corresponds to amino acids 165-184 of SEQ ID NO: 114.9C12 bound to peptide #2099 (SEQ ID NO: 522), which corresponds to aminoacids 120-139 of SEQ ID NO: 114. The other antibodies bind to regionsthat were not examined in these studies.

Subsequent to epitope mapping, the antibodies were tested by FACSanalysis on a cell line that stably expressed P503S to confirm that theantibodies bind to cell surface epitopes. Cells stably transfected witha control plasmid were employed as a negative control. Cells werestained live with no fixative. 0.5 ug of anti-P503S monoclonal antibodywas added and cells were incubated on ice for 30 min before being washedtwice and incubated with a FITC-labelled goat anti-rabbit or mousesecondary antibody for 20 min. After being washed twice, cells wereanalyzed with an Excalibur fluorescent activated cell sorter. Themonoclonal antibodies 1C3, 1D12, 9C12, 20D4 and JA1, but not 8D3, werefound to bind to a cell surface epitope of P503S.

In order to determine which tissues express P503S, immunohistochemicalanalysis was performed, essentially as described above, on a panel ofnormal tissues (prostate, adrenal, breast, cervix, colon, duodenum, gallbladder, ileum, kidney, ovary, pancreas, parotid gland, skeletal muscle,spleen and testis). HRP-labeled anti-mouse or anti-rabbit antibodyfollowed by incubation with TMB was used to visualize P503Simmunoreactivity. P503S was found to be highly expressed in prostatetissue, with lower levels of expression being observed in cervix, colon,ileum and kidney, and no expression being observed in adrenal, breast,duodenum, gall bladder, ovary, pancreas, parotid gland, skeletal muscle,spleen and testis.

Western blot analysis was used to characterize anti-P503S monoclonalantibody specificity. SDS-PAGE was performed on recombinant (rec) P503Sexpressed in and purified from bacteria and on lysates from HEK293 cellstransfected with full length P503S. Protein was transferred tonitrocellulose and then Western blotted with each of the anti-P503Smonoclonal antibodies (20D4, JA1, 1D12, 6D12 and 9C12) at an antibodyconcentration of 1 ug/ml. Protein was detected using horse radishperoxidase (HRP) conjugated to either a goat anti-mouse monoclonalantibody or to protein A-sepharose. The monoclonal antibody 20D4detected the appropriate molecular weight 14 kDa recombinant P503S(amino acids 113-241) and the 23.5 kDa species in the HEK293 celllysates transfected with full length P503S. Other anti-P503S monoclonalantibodies displayed similar specificity by Western blot.

d) Preparation and Characterization of Antibodies Against P703P

Rabbits were immunized with either a truncated (P703Ptr1; SEQ ID NO:172) or full-length mature form (P703Pfl; SEQ ID NO: 523) of recombinantP703P protein was expressed in and purified from bacteria as describedabove. Affinity purified polyclonal antibody was generated usingimmunogen P703Pfl or P703Ptr1 attached to a solid support. Rabbitmonoclonal antibodies were isolated using SLAM technology at ImmgenicsPharmaceuticals. Table VII below lists both the polyclonal andmonoclonal antibodies that were generated against P703P.

TABLE VIII Antibody Immunogen Species/type Aff. Purif. P703P(truncated); P703Ptrl Rabbit polyclonal #2594 Aff. Purif. P703P (fulllength); P703Pfl Rabbit polyclonal #9245 2D4 P703Ptrl Rabbit monoclonal8H2 P703Ptrl Rabbit monoclonal 7H8 P703Ptrl Rabbit monoclonal

The DNA sequences encoding the complementarity determining regions(CDRs) for the rabbit monoclonal antibodies 8H2, 7H8 and 2D4 weredetermined and are provided in SEQ ID NO: 506-508, respectively.

Epitope mapping studies were performed as described above. Monoclonalantibodies 2D4 and 7H8 were found to specifically bind to the peptidesof SEQ ID NO: 509 (corresponding to amino acids 145-159 of SEQ ID NO:172) and SEQ ID NO: 510 (corresponding to amino acids 11-25 of SEQ IDNO: 172), respectively. The polyclonal antibody 2594 was found to bindto the peptides of SEQ ID NO: 511-514, with the polyclonal antibody 9427binding to the peptides of SEQ ID NO: 515-517.

The specificity of the anti-P703P antibodies was determined by Westernblot analysis as follows. SDS-PAGE was performed on (1) bacteriallyexpressed recombinant antigen; (2) lysates of HEK293 cells and Ltk−/−cells either untransfected or transfected with a plasmid expressing fulllength P703P; and (3) supernatant isolated from these cell cultures.Protein was transferred to nitrocellulose and then Western blotted usingthe anti-P703P polyclonal antibody #2594 at an antibody concentration of1 ug/ml. Protein was detected using horse radish peroxidase (HRP)conjugated to an anti-rabbit antibody. A 35 kDa immunoreactive bandcould be observed with recombinant P703P. Recombinant P703P runs at aslightly higher molecular weight since it is epitope tagged. In lysatesand supernatants from cells transfected with full length P703P, a 30 kDaband corresponding to P703P was observed. To assure specificity, lysatesfrom HEK293 cells stably transfected with a control plasmid were alsotested and were negative for P703P expression. Other anti-P703Pantibodies showed similar results.

Immunohistochemical studies were performed as described above, usinganti-P703P monoclonal antibody. P703P was found to be expressed at highlevels in normal prostate and prostate tumor tissue but was notdetectable in all other tissues tested (breast tumor, lung tumor andnormal kidney).

e) Preparation and Characterization of Antibodies Against P504S

Full-length P504S (SEQ ID NO: 108) was expressed and purified frombacteria essentially as described above for P501S and employed to raiserabbit monoclonal antibodies using Selected Lymphocyte Antibody Method(SLAM) technology at Immgenics Pharmaceuticals (Vancouver, BC, Canada).The anti-P504S monoclonal antibody 13H4 was shown by Western blot tobind to both expressed recombinant P504S and to native P504S in tumorcells.

Immunohistochemical studies using 13H4 to assess P504S expression invarious prostate tissues were performed as described above. A total of104 cases, including 65 cases of radical prostatectomies with prostatecancer (PC), 26 cases of prostate biopsies and 13 cases of benignprostate hyperplasia (BPH), were stained with the anti-P504S monoclonalantibody 13H4. P504S showed strongly cytoplasmic granular staining in64/65 (98.5%) of PCs in prostatectomies and 26/26 (100%) of PCs inprostatic biopsies. P504S was stained strongly and diffusely incarcinomas (4+ in 91.2% of cases of PC; 3+ in 5.5%; 2+ in 2.2% and 1+ in1.1%) and high grade prostatic intraepithelial neoplasia (4+ in allcases). The expression of P504S did not vary with Gleason score. Only17/91 (18.7%) of cases of NP/BPH around PC and 2/13 (15.4%) of BPH caseswere focally (1+, no 2+ to 4+ in all cases) and weakly positive forP504S in large glands. Expression of P504S was not found in smallatrophic glands, postatrophic hyperplasia, basal cell hyperplasia andtransitional cell metaplasia in either biopsies or prostatectomies.P504S was thus found to be over-expressed in all Gleason scores ofprostate cancer (98.5 to 100% of sensitivity) and exhibited only focalpositivities in large normal glands in 19/104 of cases (82.3% ofspecificity). These findings indicate that P504S may be usefullyemployed for the diagnosis of prostate cancer.

Example 19 Characterization of Cell Surface Expression and ChromosomeLocalization of the Prostate-Specific Antigen P501S

This example describes studies demonstrating that the prostate-specificantigen P501S is expressed on the surface of cells, together withstudies to determine the probable chromosomal location of P501S.

The protein P501S (SEQ ID NO: 113) is predicted to have 11 transmembranedomains. Based on the discovery that the epitope recognized by theanti-P501S monoclonal antibody 10E3-G4-D3 (described above in Example17) is intracellular, it was predicted that following transmembranedeterminants would allow the prediction of extracellular domains ofP501S. FIG. 9 is a schematic representation of the P501S protein showingthe predicted location of the transmembrane domains and theintracellular epitope described in Example 17. Underlined sequencerepresents the predicted transmembrane domains, bold sequence representsthe predicted extracellular domains, and italicized sequence representsthe predicted intracellular domains. Sequence that is both bold andunderlined represents sequence employed to generate polyclonal rabbitserum. The location of the transmembrane domains was predicted usingHHMTOP as described by Tusnady and Simon (Principles Governing AminoAcid Composition of Integral Membrane Proteins: Applications to TopologyPrediction, J. Mol. Biol. 283:489-506, 1998).

Based on FIG. 9, the P501S domain flanked by the transmembrane domainscorresponding to amino acids 274-295 and 323-342 is predicted to beextracellular. The peptide of SEQ ID NO: 518 corresponds to amino acids306-320 of P501S and lies in the predicted extracellular domain. Thepeptide of SEQ ID NO: 519, which is identical to the peptide of SEQ IDNO: 518 with the exception of the substitution of the histidine with anasparginine, was synthesized as described above. A Cys-Gly was added tothe C-terminus of the peptide to facilitate conjugation to the carrierprotein. Cleavage of the peptide from the solid support was carried outusing the following cleavage mixture: trifluoroaceticacid:ethanediol:thioanisol:water:phenol (40:1:2:2:3). After cleaving fortwo hours, the peptide was precipitated in cold ether. The peptidepellet was then dissolved in 10% v/v acetic acid and lyophilized priorto purification by C18 reverse phase hplc. A gradient of 5-60%acetonitrile (containing 0.05% TFA) in water (containing 0.05% TFA) wasused to elute the peptide. The purity of the peptide was verified byhplc and mass spectrometry, and was determined to be >95%. The purifiedpeptide was used to generate rabbit polyclonal antisera as describedabove.

Surface expression of P501S was examined by FACS analysis. Cells werestained with the polyclonal anti-P501S peptide serum at 10 μg/ml,washed, incubated with a secondary FITC-conjugated goat anti-rabbit Igantibody (ICN), washed and analyzed for FITC fluorescence using anExcalibur fluorescence activated cell sorter. For FACS analysis oftransduced cells, B-LCL were retrovirally transduced with P501S. Todemonstrate specificity in these assays, B-LCL transduced with anirrelevant antigen (P703P) or nontransduced were stained in parallel.For FACS analysis of prostate tumor cell lines, Lncap, PC-3 and DU-145were utilized. Prostate tumor cell lines were dissociated from tissueculture plates using cell dissociation medium and stained as above. Allsamples were treated with propidium iodide (PI) prior to FACS analysis,and data was obtained from PI-excluding (i.e., intact andnon-permeabilized) cells. The rabbit polyclonal serum generated againstthe peptide of SEQ ID NO: 519 was shown to specifically recognize thesurface of cells transduced to express P501S, demonstrating that theepitope recognized by the polyclonal serum is extracellular.

To determine biochemically if P501S is expressed on the cell surface,peripheral membranes from Lncap cells were isolated and subjected toWestern blot analysis. Specifically, Lncap cells were lysed using adounce homogenizer in 5 ml of homogenization buffer (250 mM sucrose, 10mM HEPES, 1 mM EDTA, pH 8.0, 1 complete protease inhibitor tablet(Boehringer Mannheim)). Lysate samples were spun at 1000 g for 5 min at4° C. The supernatant was then spun at 8000 g for 10 min at 4° C.Supernatant from the 8000 g spin was recovered and subjected to a100,000 g spin for 30 min at 4° C. to recover peripheral membrane.Samples were then separated by SDS-PAGE and Western blotted with themouse monoclonal antibody 10E3-G4-D3 (described above in Example 17)using conditions described above. Recombinant purified P501S, as well asHEK293 cells transfected with and over-expressing P501S were included aspositive controls for P501S detection. LCL cell lysate was included as anegative control. P501S could be detected in Lncap total cell lysate,the 8000 g (internal membrane) fraction and also in the 100,000 g(plasma membrane) fraction. These results indicate that P501S isexpressed at, and localizes to, the peripheral membrane.

To demonstrate that the rabbit polyclonal antiserum generated to thepeptide of SEQ ID NO: 519 specifically recognizes this peptide as wellas the corresponding native peptide of SEQ ID NO: 518, ELISA analyseswere performed. For these analyses, flat-bottomed 96 well microtiterplates were coated with either the peptide of SEQ ID NO: 519, the longerpeptide of SEQ ID NO: 520 that spans the entire predicted extracellulardomain, the peptide of SEQ ID NO: 521 which represents the epitoperecognized by the P501S-specific antibody 10E3-G4-D3, or a P501Sfragment (corresponding to amino acids 355-526 of SEQ ID NO: 113) thatdoes not include the immunizing peptide sequence, at 1 μg/ml for 2 hoursat 37° C. Wells were aspirated, blocked with phosphate buffered salinecontaining 1% (w/v) BSA for 2 hours at room temperature and subsequentlywashed in PBS containing 0.1% Tween 20 (PBST). Purified anti-P501Spolyclonal rabbit serum was added at 2 fold dilutions (1000 ng-125 ng)in PBST and incubated for 30 min at room temperature. This was followedby washing 6 times with PBST and incubating with HRP-conjugated goatanti-rabbit IgG (H+L) Affinipure F(ab′) fragment at 1:20000 for 30 min.Plates were then washed and incubated for 15 min in tetramethylbenzidine. Reactions were stopped by the addition of 1N sulfuric acidand plates were read at 450 nm using an ELISA plate reader. As shown inFIG. 11, the anti-P501S polyclonal rabbit serum specifically recognizedthe peptide of SEQ ID NO: 519 used in the immunization as well as thelonger peptide of SEQ ID NO: 520, but did not recognize the irrelevantP501S-derived peptides and fragments.

In further studies, rabbits were immunized with peptides derived fromthe P501S sequence and predicted to be either extracellular orintracellular, as shown in FIG. 9. Polyclonal rabbit sera were isolatedand polyclonal antibodies in the serum were purified, as describedabove. To determine specific reactivity with P501S, FACS analysis wasemployed, utilizing either B-LCL transduced with P501S or the irrelevantantigen P703P, of B-LCL infected with vaccinia virus-expressing P501S.For surface expression, dead and non-intact cells were excluded from theanalysis as described above. For intracellular staining, cells werefixed and permeabilized as described above. Rabbit polyclonal serumgenerated against the peptide of SEQ ID NO: 548, which corresponds toamino acids 181-198 of P501S, was found to recognize a surface epitopeof P501S. Rabbit polyclonal serum generated against the peptide SEQ IDNO: 551, which corresponds to amino acids 543-553 of P501S, was found torecognize an epitope that was either potentially extracellular orintracellular since in different experiments intact or permeabilizedcells were recognized by the polyclonal sera. Based on similar deductivereasoning, the sequences of SEQ ID NO: 541-547, 549 and 550, whichcorrespond to amino acids 109-122, 539-553, 509-520, 37-54, 342-359,295-323, 217-274, 143-160 and 75-88, respectively, of P501S, can beconsidered to be potential surface epitopes of P501S recognized byantibodies.

In further studies, mouse monoclonal antibodies were raised againstamino acids 296 to 322 to P501S, which are predicted to be in anextracellular domain. A/J mice were immunized with P501S/adenovirus,followed by subsequent boosts with an E. coli recombinant protein,referred to as P501N, that contains amino acids 296 to 322 of P501S, andwith peptide 296-322 (SEQ ID NO: 898) coupled with KLH. The mice weresubsequently used for splenic B cell fusions to generate anti-peptidehybridomas. The resulting 3 clones, referred to as 4F4 (IgG1,kappa), 4G5(IgG2a,kappa) and 9B9 (IgG1,kappa), were grown for antibody production.The 4G5 mAb was purified by passing the supernatant over a ProteinA-sepharose column, followed by antibody elution using 0.2M glycine, pH2.3. Purified antibody was neutralized by the addition of 1M Tris, pH 8,and buffer exchanged into PBS.

For ELISA analysis, 96 well plates were coated with P501S peptide296-322 (referred to as P501-long), an irrelevant P775 peptide, P501S-N,P501 TR2, P501S-long-KLH, P501S peptide 306-319 (referred to asP501-short)-KLH, or the irrelevant peptide 2073-KLH, all at aconcentration of 2 ug/ml and allowed to incubate for 60 minutes at 37°C. After coating, plates were washed 5× with PBS+0.1% Tween and thenblocked with PBS, 0.5% BSA, 0.4% Tween20 for 2 hours at roomtemperature. Following the addition of supernatants or purified mAb, theplates were incubated for 60 minutes at room temperature. Plates werewashed as above and donkey anti-mouse IgHRP-linked secondary antibodywas added and incubated for 30 minutes at room temperature, followed bya final washing as above. TMB peroxidase substrate was added andincubated 15 minutes at room temperature in the dark. The reaction wasstopped by the addition of 1N H₂SO₄ and the OD was read at 450 nM. Allthree hybrid clones secreted mAb that recognized peptide 296-322 and therecombinant protein P501N.

For FACS analysis, HEK293 cells were transiently transfected with aP501S/VR1012 expression constructs using Fugene 6 reagent. After 2 daysof culture, cells were harvested and washed, then incubated withpurified 4G5 mAb for 30 minutes on ice. After several washes in PBS,0.5% BSA, 0.01% azide, goat anti-mouse Ig-FITC was added to the cellsand incubated for 30 minutes on ice. Cells were washed and resuspendedin wash buffer including 1% propidium iodide and subjected to FACSanalysis. The FACS analysis confirmed that amino acids 296-322 of P501Sare in an extracellular domain and are cell surface expressed.

The chromosomal location of P501S was determined using the GeneBridge 4Radiation Hybrid panel (Research Genetics). The PCR primers of SEQ IDNO: 528 and 529 were employed in PCR with DNA pools from the hybridpanel according to the manufacturer's directions. After 38 cycles ofamplification, the reaction products were separated on a 1.2% agarosegel, and the results were analyzed through the Whitehead Institute/MITCenter for Genome Research web server(http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) to determinethe probable chromosomal location. Using this approach, P501S was mappedto the long arm of chromosome 1 at WI-9641 between q32 and q42. Thisregion of chromosome 1 has been linked to prostate cancer susceptibilityin hereditary prostate cancer (Smith et al. Science 274:1371-1374, 1996and Berthon et al. Am. J. Hum. Genet. 62:1416-1424, 1998). These resultssuggest that P501S may play a role in prostate cancer malignancy.

Example 20 Regulation of Expression of the Prostate-Specific AntigenP501S

Steroid (androgen) hormone modulation is a common treatment modality inprostate cancer. The expression of a number of prostate tissue-specificantigens have previously been demonstrated to respond to androgen. Theresponsiveness of the prostate-specific antigen P501S to androgentreatment was examined in a tissue culture system as follows.

Cells from the prostate tumor cell line LNCaP were plated at 1.5×10⁶cells/T75 flask (for RNA isolation) or 3×10⁵ cells/well of a 6-wellplate (for FACS analysis) and grown overnight in RPMI 1640 mediacontaining 10% charcoal-stripped fetal calf serum (BRL LifeTechnologies, Gaithersburg, Md.). Cell culture was continued for anadditional 72 hours in RPMI 1640 media containing 10% charcoal-strippedfetal calf serum, with 1 nM of the synthetic androgen Methyltrienolone(R1881; New England Nuclear) added at various time points. Cells werethen harvested for RNA isolation and FACS analysis at 0, 1, 2, 4, 8, 16,24, 28 and 72-hours post androgen addition. FACS analysis was performedusing the anti-P501S antibody 10E3-G4-D3 and permeabilized cells.

For Northern analysis, 5-10 micrograms of total RNA was run on aformaldehyde denaturing gel, transferred to Hybond-N nylon membrane(Amersham Pharmacia Biotech, Piscataway, N.J.), cross-linked and stainedwith methylene blue. The filter was then prehybridized with Church'sBuffer (250 mM Na₂HPO₄, 70 mM H₃PO₄, 1 mM EDTA, 1% SDS, 1% BSA in pH7.2) at 65° C. for 1 hour. P501S DNA was labeled with 32P using HighPrime random-primed DNA labeling kit (Boehringer Mannheim).Unincorporated label was removed using MicroSpin S300-HR columns(Amersham Pharmacia Biotech). The RNA filter was then hybridized withfresh Church's Buffer containing labeled cDNA overnight, washed with1×SCP (0.1 M NaCl, 0.03 M Na₂HPO₄.7H₂O, 0.001 M Na₂EDTA), 1% sarkosyl(n-lauroylsarcosine) and exposed to X-ray film.

Using both FACS and Northern analysis, P501S message and protein levelswere found in increase in response to androgen treatment.

Example 21 Preparation of Fusion Proteins of Prostate-Specific Antigens

The example describes the preparation of a fusion protein of theprostate-specific antigen P703P and a truncated form of the knownprostate antigen PSA. The truncated form of PSA has a 21 amino aciddeletion around the active serine site. The expression construct for thefusion protein also has a restriction site at 3′ end, immediately priorto the termination codon, to aid in adding cDNA for additional antigens.

The full-length cDNA for PSA was obtained by RT-PCR from a pool of RNAfrom human prostate tumor tissues using the primers of SEQ ID NO: 607and 608, and cloned in the vector pCR-Blunt II-TOPO. The resulting cDNAwas employed as a template to make two different fragments of PSA by PCRwith two sets of primers (SEQ ID NO: 609 and 610; and SEQ ID NO: 611 and612). The PCR products having the expected size were used as templatesto make truncated forms of PSA by PCR with the primers of SEQ ID NO: 611and 613, which generated PSA (delta 208-218 in amino acids). The cDNAfor the mature form of P703P with a 6× histidine tag at the 5′ end, wasprepared by PCR with P703P and the primers of SEQ ID NO: 614 and 615.The cDNA for the fusion of P703P with the truncated form of PSA(referred to as FOPP) was then obtained by PCR using the modified P703PcDNA and the truncated form of PSA cDNA as templates and the primers ofSEQ ID NO: 614 and 615. The FOPP cDNA was cloned into the NdeI site andXhoI site of the expression vector pCRX1, and confirmed by DNAsequencing. The determined cDNA sequence for the fusion construct FOPPis provided in SEQ ID NO: 616, with the amino acid sequence beingprovided in SEQ ID NO: 617.

The fusion FOPP was expressed as a single recombinant protein in E. colias follows. The expression plasmid pCRX1 FOPP was transformed into theE. coli strain BL21-CodonPlus RIL. The transformant was shown to expressFOPP protein upon induction with 1 mM IPTG. The culture of thecorresponding expression clone was inoculated into 25 ml LB brothcontaining 50 ug/ml kanamycin and 34 ug/ml chloramphenicol, grown at 37°C. to OD600 of about 1, and stored at 4° C. overnight. The culture wasdiluted into 1 liter of TB LB containing 50 ug/ml kanamycin and 34 ug/mlchloramphenicol, and grown at 37° C. to OD600 of 0.4. IPTG was added toa final concentration of 1 mM, and the culture was incubated at 30° C.for 3 hours. The cells were pelleted by centrifugation at 5,000 RPM for8 min. To purify the protein, the cell pellet was suspended in 25 ml of10 mM Tris-Cl pH 8.0, 2 mM PMSF, complete protease inhibitor and 15 uglysozyme. The cells were lysed at 4° C. for 30 minutes, sonicatedseveral times and the lysate centrifuged for 30 minutes at 10,000×g. Theprecipitate, which contained the inclusion body, was washed twice with10 mM Tris-Cl pH 8.0 and 1% CHAPS. The inclusion body was dissolved in40 ml of 10 mM Tris-Cl pH 8.0, 100 mM sodium phosphate and 8 M urea. Thesolution was bound to 8 ml Ni-NTA (Qiagen) for one hour at roomtemperature. The mixture was poured into a 25 ml column and washed with50 ml of 10 mM Tris-Cl pH 6.3, 100 mM sodium phosphate, 0.5% DOC and 8Murea. The bound protein was eluted with 350 mM imidazole, 10 mM Tris-ClpH 8.0, 100 mM sodium phosphate and 8 M urea. The fractions containingFOPP proteins were combined and dialyzed extensively against 10 mMTris-Cl pH 4.6, aliquoted and stored at −70° C.

Example 22 Real-Time PCR Characterization of the Prostate-SpecificAntigen P501S in Peripheral Blood of Prostate Cancer Patients

Circulating epithelial cells were isolated from fresh blood of normalindividuals and metastatic prostate cancer patients, mRNA isolated andcDNA prepared using real-time PCR procedures. Real-time PCR wasperformed with the Taqman™ procedure using both gene specific primersand probes to determine the levels of gene expression.

Epithelial cells were enriched from blood samples using animmunomagnetic bead separation method (Dynal A. S., Oslo, Norway).Isolated cells were lysed and the magnetic beads removed. The lysate wasthen processed for poly A+ mRNA isolation using magnetic beads coatedwith Oligo(dT)25. After washing the beads in buffer, bead/poly A+ RNAsamples were suspended in 10 mM Tris HCl pH 8.0 and subjected toreversed transcription. The resulting cDNA was subjected to real-timePCR using gene specific primers. Beta-actin content was also determinedand used for normalization. Samples with P501S copies greater than themean of the normal samples+3 standard deviations were consideredpositive. Real time PCR on blood samples was performed using the Taqman™procedure but extending to 50 cycles using forward and reverse primersand probes specific for P501S. Of the eight samples tested, 6 werepositive for P501S and β-actin signal. The remaining 2 samples had nodetectable M-actin or P501S, No P501S signal was observed in the fournormal blood samples tested.

Example 23 Expression of the Prostate-Specific Antigens P703P and P501Sin SCID Mouse-Passaged Prostate Tumors

When considering the effectiveness of antigens in the treatment ofprostate cancer, the continued presence of the antigens in tumors duringandrogen ablation therapy is important. The presence of theprostate-specific antigens P703P and P501S in prostate tumor samplesgrown in SCID mice in the presence of testosterone was evaluated asfollows.

Two prostate tumors that had metastasized to the bone were removed frompatients, implanted into SCID mice and grown in the presence oftestosterone. Tumors were evaluated for mRNA expression of P703P, P501Sand PSA using quantitative real time PCR with the SYBR green assaymethod. Expression of P703P and P501S in a prostate tumor was used as apositive control and the absence in normal intestine and normal heart asnegative controls. In both cases, the specific mRNA was present in latepassage tumors. Since the bone metastases were grown in the presence oftestosterone, this implies that the presence of these genes would not belost during androgen ablation therapy.

Example 24 Identification of P501S and P703P-Specific Antibodies inProstate Cancer Patients

Antibody responses specific for the prostate antigens P501S or P703P insera of prostate cancer patients and normal healthy donors were examinedas follows.

Both Western blot analysis and ELISA analysis of sera were performedusing P501S peptides (SEQ ID NO: 985-987) and P703P peptides (SEQ ID NO:988-990). SEQ ID NO: 985, 986 and 987 represent amino acids 231-250,246-266 and 492-521, respectively, of P501S. SEQ ID NO: 988, 989, 990,1030, 1031, 1032 1033, and 1037 represent amino acids 110-124, 140-154,190-204, 62-76, 91-106, 200-214, and 229-245 respectively, of P703P. Theresults demonstrated that some of the prostate cancer patients hadantibodies that recognize the same peptides (i.e. common epitopes) ofeither P501S or P703P and that these common epitopes were either notrecognized or recognized at significantly lower levels by sera fromnormal donors. The data showed that at least 14 out of 48 prostatecancer patients possessed P501S-specific antibodies, and that 3 out of48 prostate cancer patients possessed P703P-specific antibodies withnone of the patients having both antibodies specific for P501S andantibodies specific for P703P.

Example 25 Anti-P503S Monoclonal Antibody Inhibits Tumor Growth In Vivo

The ability of the anti-P503S monoclonal antibody 20D4 to suppress tumorformation in mice was examined as follows.

Ten SCID mice were injected subcutaneously with HEK293 cells thatexpressed P503S. Five mice received 150 micrograms of 20D4 intravenouslyat day 0 (time of tumor cell injection), day 5 and day 9. Tumor size wasmeasured for 50 days. Of the five animals that received no 20D4, threeformed detectable tumors after about 2 weeks which continued to enlargethroughout the study. In contrast, none of the five mice that received20D4 formed tumors. These results demonstrate that the anti-P503S Mab20D4 displays potent anti-tumor activity in vivo.

Example 26 Characterization of a T Cell Receptor Clone from aP501S-Specific T Cell Clone

T cells have a limited lifespan. However, cloning of T cell receptor(TCR) chains and subsequent transfer essentially enables infinitepropagation of the T cell specificity. Cloning of tumor-antigen TCRchains allows the transfer of the specificity into T cells isolated frompatients that share the TCR MHC-restricting allele. Such T cells couldthen be expanded and used in adoptive transfer settings to introduce thetumor antigen specificity into patients carrying tumors that express theantigen. T cell receptor alpha and beta chains from a CD8 T cell clonespecific for the prostate-specific antigen P501S were isolated andsequenced as follows.

Total mRNA from 2×10⁶ cells from CTL clone 4E5 (described above inExample 12) was isolated using Trizol reagent and cDNA was synthesized.To determine Va and Vb sequences in this clone, a panel of Va and Vbsubtype-specific primers was synthesized and used in RT-PCR reactionswith cDNA generated from each of the clones. The RT-PCR reactionsdemonstrated that each of the clones expressed a common Vb sequence thatcorresponded to the Vb7 subfamily. Furthermore, using cDNA generatedfrom the clone, the Va sequence expressed was determined to be Va6. Toclone the full TCR alpha and beta chains from clone 4E5, primers weredesigned that spanned the initiator and terminator-coding TCRnucleotides. The primers were as follows: TCR Valpha-6 5′(sense):GGATCC-GCCGCCACC-ATGTCACTTTCTAGCCTGCT (SEQ ID NO: 899) BamHI site KozakTCR alpha sequence TCR alpha 3′ (antisense):GTCGAC-TCAGCTGGACCACAGCCGCAG (SEQ ID NO: 900) SalI site TCR alphaconstant sequence TCR Vbeta-7. 5′(sense):GGATCC-GCCGCCACC-ATGGGCTGCAGGCTGCTCT (SEQ ID NO: 901) BamHI site KozakTCR alpha sequence TCR beta 3′ (antisense): GTCGAC-TCAGAAATCCTTTCTCTTGAC(SEQ ID NO: 902) SalI site TCR beta constant sequence. Standard 35 cycleRT-PCR reactions were established using cDNA synthesized from the CTLclone and the above primers, employing the proofreading thermostablepolymerase PWO (Roche, Nutley, N.J.).

The resultant specific bands (approx. 850 bp for alpha and approx. 950for beta) were ligated into the PCR blunt vector (Invitrogen) andtransformed into E. coli. E. coli transformed with plasmids containingfull-length alpha and beta chains were identified, and large scalepreparations of the corresponding plasmids were generated. Plasmidscontaining full-length TCR alpha and beta chains were submitted forsequencing. The sequencing reactions demonstrated the cloning offull-length TCR alpha and beta chains with the determined cDNA sequencesfor the Vb and Va chains being shown in SEQ ID NO: 903 and 904,respectively. The corresponding amino acid sequences are shown in SEQ IDNO: 905 and 906, respectively. The Va sequence was shown by nucleotidesequence alignment to be 99% identical (347/348) to Va6.2, and the Vb tobe 99% identical to Vb7 (336/338).

Example 27 Capture of Prostate Specific Cells Using the Prostate AntigenP503S

As described above, P503S is found on the surface of prostate cells.Secondary coated microsphere beads specific for mouse IgG were coupledwith the purified P503S-specific monoclonal antibody 1D12. The boundP503S antibody was then used to capture HEK cells expressing recombinantP503S. This provides a model system for prostate-specific cell capturewhich may be usefully employed in the detection of prostate cells inblood, and therefore in the detection of prostate cancer.

P503S-transfected HEK cells were harvested and redissolved in washbuffer (PBS, 0.1% BSA, 0.6% sodium citrate) at an appropriate volume togive at least 54 cells per sample. Round bottom Eppendorf tubes wereused for all procedures involving beads. The stock concentrations wereas shown below in Table IX.

TABLE IX Stock concentrations Sample concentration Amount neededEpithelial enrich beads 1⁷ beads/ml 125 ul stock per 5 ml 4⁸ beads/ml(Dynal volume Biotech Inc. Lake Success, NY) 1D12 ascites antibody 0.1ug/ml (0.1X) to 5 0.05 ul to 2.5 ul stock 2 mg/ml ug/ml (5X) titrationsper sample α-Mamma Mu 1 ug/ml (1X) 1.1 ul stock per 0.9 mg/ml samplePan-mouse IgG beads 1⁷ beads/ml 125 ul stock per 5 ml 4⁸ beads/ml (Dynalvolume Biotech)

Blocked immunomagnetic beads were pre-washed as follows: all beadsneeded were pooled and washed once with 1 ml wash buffer. The beads wereresuspended in a 3× volume of 1% BSA (v/v) in wash buffer and incubatedfor 15 min rotating at 4° C. The beads were then washed three times with2× volume of wash buffer and resuspended to original volume. Non-blockedbeads were pooled, washed three times with 2× volume of wash buffer andresuspended to original volume.

Primary antibody was incubated with secondary beads in a fresh Eppendorffor 30 minutes, rotating at 4° C. Approximately 200 ul wash buffer wasadded to increase the total volume for even mixing of the sample. Theantibody-bead solution was transferred to a fresh Eppendorf, washedtwice with an equal volume of wash buffer and resuspended to originalvolume. Target cells were added to each sample and incubated for 45minutes, rotating at 4° C. The tubes were transferred to a magnet, thesupernatant removed, taking care not the agitate the beads, and thesamples were washed twice with 1 ml wash buffer. The samples were thenready for RT-PCR using a Dynabeads mRNA direct microkit (Dynal Biotech).

Epithelial cell enrichment was placed in a magnet and supernant wasremoved. The epithelial enrichment beads were then resuspended in 100 ullysis/binding buffer fortified with Rnasin (2 U/ul per sample), andstored at −70° C. until use. Oligo (dT₂₅) Dynabeads were pre-washed asfollows: all beads needed were pooled (23 ul/sample), washed three timeswith an excess volume of lysis/binding buffer, and resuspended tooriginal volume. The lysis supernant was separated with a magnet andtransferred to a fresh Eppendorf. 20 ul oligo(dT25) Dynabeads were addedper sample and rolled for 5 min at room temperature. Supernant wasseparated using a magnet and discarded, leaving the mRNA annealed to thebeads. The bead/mRNA complex was washed with buffer and resuspended incold Tris-HCl.

For RT-PCR, the Tris-HCl supernatant was separated and discarded usingMPS. For each sample containing 1⁵ cells or less, the following wasadded to give a total volume of 30 ul: 14.25 ul H₂O; 1.5 ul BSA; 6 ulfirst strand buffer; 0.75 mL 10 mM dNTP mix; 3 ul Rnasin; 3 ul 0.1M dTT;and 1.5 ul Superscript II. The resulting solution was incubated for 1hour at 42° C., diluted 1:5 in H2O, heated at 80° C. for 2 min to detachcDNA from the beads, and immediately placed on MPS. The supernatantcontaining cDNA was transferred to a new tube and stored at −20° C.

Table X shows the percentage of capture of P503S-transfected HEK cellsas determined by RT-PCR.

TABLE X % capture P503S- % capture transfected HEK cells LnCAP cells 0.1ug/ml P503S Mab 36.90 0.00 0.5 ug/ml P503S Mab 67.40 2.93 1 ug/ml P503SMab 40.22 0.00 5 ug/ml P503S Mab 13.11 0.00 Anti-Mu beads only, 1.420.00 non-blocked Anti-Mu beads only, 15.65 20.21 blocked Absolutecontrol, non- 100.00 100.00 capture cells

Example 28 Immunization of Mice with Recombinant P703P

In vivo immunogenicity studies were performed using a variety of P703Precombinant protein formulations. Specifically, groups of mice wereimmunized with the P703P formulations shown below in Table XI, wherein“C'amidated P703P” represents P703P amidated at the C terminal;“truncated-P703P” represents a truncated form of P703P, and “FOPP”represents a fusion of P703P and PSA.

TABLE XI GROUP ANTIGEN DOSE SOURCE ADJUVANT ROUTE 1 C′amidated 20 ug E.coli AS1 Im, sq (fp) P703P 2 Truncated 20 ug E. coli AS1 Im, sq (fp)P703P 3 C′amidated 20 ug Pichia AS1 Im, sq (fp) P703P 4 P703P 20 ugPichia AS1 Im, sq (fp) 5 FOPP 20 ug E. coli AS1 Im, sq (fp) 6 P703P 10⁷pfu none Sq base of tail 7 C′amidated 20 ug E. coli MPL-SE Im, sq (fp)P703P 8 Truncated 20 ug E. coli MPL-SE Im, sq (fp) P703P 9 C′amidated 20ug Pichia MPL-SE Im, sq (fp) P703P 10 P703P 20 ug Pichia MPL-SE Im, sq(fp) 11 FOPP 20 ug E. coli MPL-SE Im, sq (fp) 12 Naïve (control)

Each protein immunization was done in four sites: subcutaneously (sq) inboth footpads and intramuscularly (im) in the leg. Each immunization wasdone 3 weeks apart, with sera plus spleen and lymph node (LN) cellsbeing harvested 10 days following the last immunization.

T cell proliferation and interferon-gamma assays were performed asfollows. 250,000 spleen or 100,000 LN cells were plated in 96 wellplates and stimulated with 1-10 ug/ml of antigen. Antigens testedinclude the five proteins listed above, P703P expressed in baculovirus,NS1 control protein, PSA (for FOPP groups only), and P703P peptidepools. Peptide pools consisted of 20-mer peptides overlapping by 15amino acids with each pool containing 6-8 peptides. Con A was used as apositive control. Cultures were pulsed with H3-thymidine on day 4 afterinitiation of culture for assaying proliferation. For assaying IFNγlevels by ELISA, supernatants were also pulled on day 4. In addition,sera were pooled and assayed by ELISA for IgG antibodies against therecombinant proteins listed above.

All immunogens elicited strong antibody responses to the immunogen. Inall cases these responses reacted with other sources of P703P protein,including strong reactivity with both Pichia forms and baculovirus formsof P703P. AS1 adjuvant elicited stronger antibody responses than MPL-SE.The best immunogen in terms of eliciting a response against Pichia andbaculovirus forms of P703P was FOPP, but all of the immunogens elicitedstrong reactive P703P antibody responses against the E. coli derivedP703P. In both the proliferation and interferon-gamma assays, allimmunogens elicited fairly good T cell response to the immunogen, withmost animals with detectable responses to their immunogen alsoresponding to other sources of P703P protein. Again, AS1 adjuvantelicited better T cell responses than MPL-SE.

Example 29 Analysis of P703P Expression in Prostate Cancer and NormalTissue by Immunohistochemistry (IHC)

Protein expression of the prostate cancer antigen P703P was analyzed inprostate cancer and normal tissue by IHC analysis using an array ofanti-P703P polyclonal and monoclonal antibodies (see Table XII). P703Pprotein expression was also analyzed in HEK293/P703P transfectants byflow cytometry and by Western blot analysis using the same anti-P703Pantibodies. The P703-R087 and P703P-2059 antibodies stained HEK293/P703Ptransfectants by flow cytometric analysis. P703P-2594 was used forWestern blot analysis.

TABLE XII ANTI-P703 ANTIBODIES Antibody Lot# Species/type ImmunogenPurification P703P-2594 370-84  Rabbit bacteria derived Immunogenpolyclonal truncated P703P (c- term 159 aa) P703P-9245 690.46 Rabbitbacteria derived full Immunogen polyclonal length P703/NS1 fusionP703-2D4 Rabbit bacteria derived Protein A polyclonal truncated P703P(c- term 159 aa) P703-8H2 Rabbit bacteria derived Protein A monoclonaltruncated P703P (c- term 159 aa) P703-7H8 Rabbit bacteria derivedProtein A monoclonal truncated P703P (c- term 159 aa) P703P-2059 690.88Rabbit Baulovirus derived Immunogen polyclonal full length form P703P-Rabbit Baulovirus derived Protein A R087 monoclonal full length formP703P- Rabbit Baulovirus derived Protein A R080 monoclonal full lengthform PA-P703P- 858-130 Rabbit bacteria derived Protein A 9245 polyclonalfull length P703/ NS1 fusion AP-P703P- 858-109 Rabbit bacteria derivedBaulovirus 9245 polyclonal fulll ength P703/ full length NS1 fusion

In order to further evaluate tissues expression of the prostate cancerantigen P703P protein, immunohistochemistry (IHC) analysis was performedon a diverse range of tissue sections. Tissue samples were fixed informalin solution for 12-24 hrs and embedded in paraffin before beingsliced into 8 micron sections. Steam heat induced epitope retrieval(SHIER) in 0.1 M sodium citrate buffer (pH 6.0) was used for optimalstaining conditions. Sections were incubated with 10% serum/PBS for 5minutes. Primary antibody was added to each section for 25 minutes atindicated concentrations followed by 25 minute incubation withanti-rabbit biotinylated antibody. Endogenous peroxidase activity wasblocked by three 1.5 minute incubations with hydrogen peroxidase. Theavidin biotin complex/horse radish peroxidase (ABC/HRP) system was usedalong with DAB chromogen to visualize antigen expression. Slides werecounterstained with hematoxylin to visualize cell nuclei. For Westernblot analysis, whole cell lysates were generated by incubating the cellsin Triton-X 100 containing lysis buffer for 30 minutes on ice. Lysateswere then cleared by centrifugation at 10,000 rpm for 5 minutes at 4° C.Samples were diluted with SDS-PAGE loading buffer containingbeta-mercaptoethanol, then boiled for 10 minutes prior to loading theSDS-PAGE gel. Protein was transferred to nitrocellulose and probed using1 ug/ml purified anti-P703P rabbit polyclonal sera 2594 (lot #370-84) orrabbit monoclonal P703P-R087. Blots were revealed using goat anti-rabbitIg coupled to HRP followed by incubation in ECL substrate. For FACSanalysis, single cell suspensions of P703P/HEK293 transfectants weregenerated and cells were incubated in permeabilization buffer(PBS+saponin+BSA) at room temp. The cells were then incubated for 30minutes with 10 ug/ml of purified anti-P703P polyclonal sera ormonoclonal antibodies. Following 3 washes in permeabilization buffer,cells were then incubated with a 1:100 dilution of goat anti-rabbitIg(H+L)-FITC(Southern Biotechnology) for 30 minutes at RT. After 3washes, the cells were resuspended in buffer and analyzed by FACS.

Optimal IHC results were obtained using the heat retrieval and affinitypurified AP-P703P-9245 antibody against full-length P703. IHC analysisusing the above procedures with this antibody showed weak staining in6/23 metastatic prostate cancer sections. 24/33 primary prostate cancersections stained positive with this antibody. Additionally, staining wasobserved in malignant ducts, benign (normal ducts) in various prostatetumors, basal, myoepithelial cells in various prostate tumors, mastcells, and in association with lipofuchsin granules.

A panel of normal tissue including brain, breast, uterus, parotid,spleen, liver, ovary, adrenal, cervix, tonsil, esophagus, bronchus,lung, kidney, testis, pancreas, skeletal muscle, colon, small intestine,skin, and thyroid sections, were all negative.

Example 30 Detection of Circulating Prostate Tumor Cells in PeripheralBlood Using Cell Capture Technology

The peripheral bloods of 55 urology patients, diagnosed with prostatecancer, benign prostatic hyperplasia (BPH), bladder and other prostatedisorders, were collected and processed using the Dynal EpithelialEnrich kit (Dynal, Lake Success, N.Y.), which uses magnetic beads coatedwith the epithelial-specific antibody Ber-EP4 to specifically capturemetastatic tumor cells. Total RNA was then isolated using the QIAGENRNeasy Mini Kit (QIAGEN, Valencia, Calif.), DNase treated, and firststrand cDNA was synthesized from the cell capture fractions using LifeTechnologies Superscript II Rnase H reverse transcriptase (InvitrogenLife Technologies, Carlsbad, Calif.). Real-time PCR was performed toevaluate the level of expression of prostate tumor genes P501S (alsoreferred to herein as L1-12, e.g., SEQ ID NO: 110), P703P (e.g., SEQ IDNO:524), P503S (also referred to herein as N1-1862, e.g., SEQ ID NO:11),P504S (also referred to herein as F1-12, e.g., SEQ ID NO:107), P510S(e.g., SEQ ID NO:535 and 536), and P1020C (also referred to as 22553,e.g., SEQ ID NO:591) using the housekeeping genes, Actin,Beta-2-Microglobulin, and Cytokeratin 19, as controls.

By combining the expression patterns of P501S and P504S, we were able todetect at least two-fold overexpression of the two genes combined for10/24 prostate cancer patients and 4/25 BPH patients as compared to themean signal for the bladder and other prostate disorders group. At leasttwo-fold mean overexpression was also detected in the cancer group ascompared to the BPH and Bladder cancer/other prostate disorders groupfor genes P501S, P503S, P703P, P504S, and P510S. In addition, the samesamples were identified with P501S, P504S, and P510S. Thus, expressionanalysis of prostate tumor sequences identified herein in blood samplesfollowing cell capture can be utilized as a diagnostic tool, for examplein the detection of circulating tumor cells, e.g., metastatic tumorcells, in patients.

Example 31 Identification of a P703P-Derived CD4+ T Cell Epitope

A total of 40 15-mer peptides overlapping by 10 amino acids spanningamino acids 47 to 254 of the P703P protein sequence were generated bystandard procedure. Dendritic cells (DC) were derived from PBMC of anormal female donor using GMCSF and IL-4 by standard procedures. CD4 Tcells were generated from the same donor as the DC using MACS beads andnegative selection. DC were pulsed overnight with pools containing 10 ofthe 15 mer peptides, with each peptide at 0.25 microgram/ml finalconcentration. Pulsed DC were washed and plated at 1×10e4 cells/well of96-well round bottom plates, and purified CD4 T cells were added at1×10e5/well. Cultures were supplemented with 60 ng/ml IL-6 and 10 ng/mlIL-12 and incubated at 37° C. Cultures were re-stimulated as above on aweekly basis and were tested for specific proliferation using as APC, DCgenerated and pulsed as above, supplemented with 5 ng/ml IL-7 and 10u/ml IL-2. Following 5 in-vitro stimulation cycles, lines (each linecorresponds to one well) were assayed for proliferation and cytokineproduction in response to the stimulating pools vs an irrelevant pool ofpeptides derived from the P703P sequence. 12 lines were identified thatdemonstrated specific proliferation (measured in standard tritiatedthymidine proliferation assays) and cytokine production (measured inγ-interferon ELISA assays) in response to the stimulating peptide pool.These lines were further tested for specific recognition of the peptidepool, specific recognition of individual peptides in the pool andspecific recognition of recombinant P703P protein. Using this approach,a peptide epitope derived from the prostate tumor specific protein,P703P, recognized by CD4+ T cells in the context of MHC class II, wasidentified. The peptide corresponds to amino acids 145-159 of the P703Pprotein sequence of SEQ ID NO: 525, and has the sequence GNSCLVSGWGLLANG(SEQ ID NO:992). The cDNA sequence encoding this epitope is set forth inSEQ ID NO:991.

Example 33 Identification of P706P Extended Sequences

Related clones were identified above in Example 1 (e.g., SEQ ID NO: 46,212, 213, 214, 215, 288 and 415) as having prostate-specific expressionpatterns. Upon further analysis, an extended sequence for these cloneswas identified as follows. Sequence specific primers were used toamplify products from a prostate tumor library, resulting in theidentification of clones 8B6-P3 (SEQ ID NO: 993) and 8B6-P4 (SEQ ID NO:994). Both clones were transformed into appropriate cells, isolated,sequenced, and the resulting sequences were used as queries to searchagainst the Genbank sequence database. This search revealed that 8B6-P3and 8B6-P4 and other related EST sequences were components of the samegenomic 7q21 BAC clone. Based on the close proximity along the genomicbackbone and on the similar expression profiles, these components wereconcluded to be part of the same gene, referred to herein as P706P.Northern analysis demonstrated high over-expression in prostate tumorsand prostate tissues, and also showed expression in brain and ovary. Thefour sequences disclosed herein (SEQ ID NO: 993-997) are representativesof the 4 longest contiguous pieces of cDNA-derived sequence along thegenomic backbone. Contigation of clones 8B6.P3 (SEQ ID NO: 993) and8-B6.P4 (SEQ ID NO: 994), using also available EST sequences, allowedthe identification of the extended P706P sequence set forth in SEQ IDNO: 995.

Example 34 Identification of P713P Extended Sequence

The identification of SEQ ID NO: 146, also referred to as P219, wasdescribed above. This example describes the identification of anextended sequence for this clone, as well as the bioinformaticidentification of the full length cDNA and protein sequences containingthe same. Full length laboratory cloning efforts were initiated with anEST search and alignment. The sequence identified by this approach wasconfirmed with a 5′ RACE assay (Clonetech; see also Chenchik et al.,1995, 1996) in which a library of adaptor ligated prostate tumor cDNAsare used as a template to amplify the desired 5′ end of a gene using the5′ adaptor primer and a gene specific 3′ primer. Additional sequence wasidentified using a nucleic acid-based screen in which a phagemidprostate tumor library was screened by growing plaques, transferring andimmobilizing DNA onto nylon filters, and hybridizing with a radioactivegene-specific probe. The filters were then exposed to film and plaquescorresponding to radioactively exposed positive hits were picked andprepped for DNA isolation. The DNA sequence identified using the aboveapproach both confirmed the original clone sequence and further yieldedadditional 5′ sequence. This extended cDNA sequence, also referred to asP713P, is set forth in SEQ ID NO: 998. In a separate analysis, it wasdetermined that P713P corresponds to a protein referred to as ProstaticSecretory Protein (PSP), the full length cDNA sequence of which is setforth in SEQ ID NO: 999, encoding the amino acid sequence set forth inSEQ ID NO: 1000.

Example 35 Identification of P1E Extended Sequence

The identification of the cDNA sequences set forth in SEQ ID NO: 620,621 and 622, also referred to as P1E, were described above in Example 1.These sequences resulted from a serological expression cloning effortand subsequent nucleic acid screening of the same cDNA expressionlibrary. An expression cDNA library was constructed from prostate tumorextracted mRNA using routine methodologies. This library was screenedwith autologous patient sera. (prostate tumor and patient sera were fromthe same individual) 10,000 pfu were plated on 6 large LB agar plates,allowed to grow up for 6 hrs, and IPTG-infused filters were applied toinduce expression and immobilize proteins as they were produced. Thefilters were oriented to the plate and were removed 12 hours later. Thefilters were blocked in PBS 1% tween, and then incubated with thepatient sera for 12 hours at 4 degrees Celsius. The washed filters wereincubated in I-125 conjugated protein A for 1 hour at room temperature,washed again and exposed to film overnight. Exposed spots on the filmindicating expressed protein recognized by the autologous sera werepicked, purified, the plasmid excised, and prepped for DNA isolation andsubsequent sequencing. This procedure resulted in the identification ofclone PT44A22.

Primers were designed from the PT44A22 sequence in order to furtherscreen the same expression library. For probe labeling, PCRamplification of the original PT4A22 DNA fragment was performed in thepresence of radioactive nucleotides. The probe was incubated with nylonfilters onto which the DNA of 10000-15000 clones of the expressionlibrary were immobilized. After a few hours of hybridization at 65degrees in Church's hybridization buffer, the filters were washed in asolution of 1×SCP, 1% sarkosyl. Film was exposed to the washed filtersovernight and positive signals were used to pick the appropriatecolonies. These picks, which contained about 50 colonies each, werefurther purified by the method described previously, resulting in a purecolony pick which was then grown up and prepped for plasmid DNA. Theplasmid containing the clone of interest was digested for fragment sizeand the insert was sequenced using flanking promoter primers. Thisprocedure resulted in the clone PT44.P2. Together, PT44A22 and PT44.P2make up the extended P1E sequence disclosed herein as SEQ ID NO: 1004,encoding the amino acid sequence set forth in SEQ ID NO: 1005.

Example 36 Immunohistochemical Analysis of P510S

Identification of the prostate specific gene P510S is described above(e.g., Example 1). In order to evaluate P510S protein expression,immunohistochemistry (IHC) analysis was performed on a diverse range oftissue sections. Tissue samples were fixed in formalin solution for12-24 hours and embedded in paraffin before being sliced into 8 micronsections. Steam heat induced epitope retrieval (SHIER) in 0.1 M sodiumcitrate buffer (pH 6.0) was used for optimal staining conditions.Sections were incubated with 10% serum/PBS for 5 minutes. Primaryantibody was added to each section for 25 minutes at indicatedconcentrations followed by a 25 minute incubation with anti-rabbitbiotinylated antibody. Endogenous peroxidase activity was blocked bythree 1.5 minute incubations with hydrogen peroxidase. The avidin biotincomplex/horse radish peroxidase (ABC/HRP) system was used along with DABchromogen to visualize antigen expression. Slides were counterstainedwith hematoxylin to visualize cell nuclei.

Using this approach, P510S protein expression was detected in 31/32primary prostate tumor samples and in 8/17 bone metastasis samples. Someexpression was also detected in 4/5 normal prostate samples and 3/5testis samples. No expression was detected in thyroid, spleen, uterus,ovary, pancreas, lung, liver, kidney, heart, stomach, large intestine,small intestine, brain and adrenal gland.

Example 37 Diagnostic Evaluation of Prostate Antigen P504S

Identification of the prostate specific sequence P504S has beendescribed above (e.g., Example 1). In order to evaluate P504S proteinexpression, immunohistochemistry (IHC) analysis was performed on adiverse range of tissue sections. Tissue samples were fixed in formalinsolution for 12-24 hours and embedded in paraffin before being slicedinto 8 micron sections. Steam heat induced epitope retrieval (SHIER) in0.1 M sodium citrate buffer (pH 6.0) was used for optimal stainingconditions. Sections were incubated with 10% serum/PBS for 5 minutes.Primary antibody was added to each section for 25 minutes at indicatedconcentrations followed by a 25 minute incubation with anti-rabbitbiotinylated antibody. Endogenous peroxidase activity was blocked bythree 1.5 minute incubations with hydrogen peroxidase. The avidin biotincomplex/horse radish peroxidase (ABC/HRP) system was used along with DABchromogen to visualize antigen expression. Slides were counterstainedwith hematoxylin to visualize cell nuclei.

A. P504S Expression in Colon Carcinomas: Correlation with Tumor Gradeand Patient Survival

This example evaluates the immunoreactivity of P504 is in normal colonand invasive colon carcinomas, and also evaluates the correlationbetween P504 expression and tumor grade, stage, and patient survival.Routinely processed resected colon carcinomas (n=44), villous adenomas(V, n=4) and tubular or tubulovillous adenomas (TA/TVA, n=24) were cutat 5 μm and immunostained with P504 on a TechMate 1000 (Ventana MedicalSystems, Tucson, Ariz.) automated immunostainer using an avidin/biotincomplex (ABC) staining procedure. Positive immunostaining in cytoplasmwas graded: 0=negative staining; 1+=up to 33% cells positive; 2+=34-66%;3+=>66%. Tumors were graded on review of H&E stained slides, and stage,location, and follow up were obtained from pathology reports and patientcharts.

Invasive carcinomas were from 18 males and 26 females, ages 45-87, fromright colon (23), transverse (6), left (14), and unknown (1). Tumorstage was I (4), II (17), III (18), and IV (5). Tumor grade was Grade 1(23), 2 (9), and 3 (12). Remnant polyp was present in 7 carcinomas.Immunostaining for p504S was weakly positive in surface epithelium ofnormal colon. In carcinomas, P504 was graded 0 (16), 1+ (6), 2+ (8), and3+ (14). There was no difference between superficial and deep regions ofthe tumors. Remnant polyp was 2+ or 3+ in 4 cases, 0 in 3 cases. Thepercentage of carcinomas positive for each grade of P504S correlatedinversely with tumor grade and patient survival. There was nocorrelation between P504S expression and TNM stage or overall stage.Most adenomas (25/35) were 2+ or 3+, regardless of histologic type.

Thus, P504S was highly expressed in adenomas and well-differentiatedcolon carcinomas, and weak or absent in normal colon mucosa and poorlydifferentiated carcinomas. P504S expression correlated inversely withpatient survival. In view of these results, immunostaining for P504Srepresents a useful adjunct with prognostic significance in thediagnostic evaluation of colon carcinomas.

B. Expression of P504S in Atypical Adenomatous Hyperplasia of theProstate

Atypical Adenomatous Hyperplasia (AAH), also known as adenosis, ischaracterized by the abnormal architectural patterns but withoutsignificant cytologic atypia in prostatic glandular epithelium. AAH mayrepresent a precursor lesion of prostatic adenocarcinoma. P504S has beenfound to be highly expressed in prostatic adenocarcinoma, but not inbenign prostatic epithelium. In this example, the expression of P504Swas evaluated in AAH.

A total of 46 cases including 26 of AAH (prostatectomy: N=18, biopsy:N=6, and transurethral resection: N=2), 10 cases of adenocarcinomas withGleason score 2+2=4 and 10 cases of benign prostatic hyperplasia (BPH)were examined by immunohistochemistry for P504S and high molecularweight cytokeratin 34βE12. 34βE12 staining confirmed the presence ofpatchy basal cells in all 26 cases of AAH. P504S showed strongcytoplasmic granular staining in all (10 of 10) low-grade carcinomas andin 4 of 26 cases of AAH (15%). No BPHs or benign glands adjacent tocarcinomas and AAH were positive for P504S. Diffuse P504S stainingpattern was found in low-grade carcinomas, while focal P504s positivitywas seen in AAH.

Thus, expression of P504S was found in all low-grade carcinomas and asmall percentage of AAH, but not in BPH or other benign glands. Thesefindings suggest that AAH is a lesion different from low-grade cancer orBPH, at least with respect to P504S expression. Moreover, AAH may beconsidered as a premalignant or potentially premalignant lesion.

C. P504S as a Diagnostic Marker for Minimal Prostatic Adenocarcinoma onNeedle Biopsy

Establishing diagnosis of minimal prostatic cancer (PC) on needle biopsyis a major diagnostic challenge. A marker specific for prostate cancerwill enhance our ability to detect minimal disease. P504S, identified bycDNA subtraction and microarray technology, has been demonstrated to behighly expressed in PC but not in benign prostatic glands. The aim ofthis study was to evaluate P504S expression as a means for detectingminimal PC.

A total of 111 prostate needle biopsies, including 52 of minimalprostatic adenocarcinoma with Gleason Score 6 (N=51) or 8 (N=1) and 59benign prostates, were examined by using immunohistochemistry specificfor P504S and high molecular weight cytokeratin (34βE12). Minimal PC,defined as a tumor focus <1 mm in diameter on H&E staining, was presentas only one single focus in the entire biopsy (N=35), or as one smallfocus in one needle core in addition to carcinoma present elsewhere(N=18).

P504S immunopositivity was found in 51/52 cases (98%) of minimal PC butnot in any benign prostates (0/58) or benign glands adjacent tomalignant glands. Atrophy, basal cell hyperplasia and transitional cellmetaplasia were all negative for P504S. The only case of PC withnegative staining of P504S showed features of foamy carcinoma in abiopsy obtained from the transition zone. High molecular weightcytokeratin (34βE12) immunostaining confirmed the absence of basal cellsin the focus of carcinoma in all 52 cases.

The high specificity and sensitivity of P504S for detecting limitedprostatic adenocarcinoma illustrates its diagnostic value in a clinicalsetting. Using P504S as a immunohistochemical marker can thus increasethe accuracy in pathology diagnosis of minimal prostatic adenocarcinomaon needle biopsy.

Example 38 Immunohistochemical Analysis of P767P

In order to evaluate protein expression of the prostate antigen P767P,immunohistochemistry (IHC) analysis was performed on a diverse range oftissue sections. Tissue samples were fixed in formalin solution for12-24 hours and embedded in paraffin before being sliced into 8 micronsections. Steam heat induced epitope retrieval (SHIER) in 0.1 M sodiumcitrate buffer (pH 6.0) was used for optimal staining conditions.Sections were incubated with 10% serum/PBS for 5 minutes. Primaryantibody was added to each section for 25 minutes at indicatedconcentrations followed by a 25 minute incubation with anti-rabbitbiotinylated antibody. Endogenous peroxidase activity was blocked bythree 1.5 minute incubations with hydrogen peroxidase. The avidin biotincomplex/horse radish peroxidase (ABC/HRP) system was used along with DABchromogen to visualize antigen expression. Slides were counterstainedwith hematoxylin to visualize cell nuclei. Using this approach, P767Sprotein expression was detected in 7/7 prostate cancers, 5/5 normalprostates, 1/1 normal kidney, 1/1 cerebellum, 0/1 normal liver, 0/1normal heart, 1/1 normal lung, 1/1 normal colon, 1/1 normal tonsil, and1/1 normal cortex.

Example 39 P510S and MAPS-P510S Constructs for DNA Immunization andAntibody Generation

The identification of prostate sequence P510S has been identified above(e.g., Example 1). Plasmid DNA containing P510S was used as template ina PCR reaction to amplify the open reading frame of P510S usingP510S-AW186 sense primers with Hind III sites and a Kozak sequence, andP510S-AW187 antisense primers with Nhe I sites and a stop codon. PCRproduct was purified from agarose gel, digested with Hind III and Nhe I,and ligated to JA4304 vector that had been linearized with the same tworestriction enzymes. The ligation mixture was transformed into E. coliand several clones were randomly selected for restriction enzymeanalysis and DNA sequencing. P510S/JA4304#2 and #3 clones were confirmedto have the desired P510S sequence. The determined DNA sequence for thisconstruct is set forth in SEQ ID NO: 1006, encoding the polypeptide ofSEQ ID NO: 1009.

For construction of MAPS-P510S, the pCRX4 vector was used. pCRX4 is aMAPS fusion vector based on JA4304 that was made to facilitate thecloning of MAPS fusion constructs. In pCRX4, MAPS was cloned down streamof Kozak sequence and was followed by a multiple cloning site. Togenerate the MAPS-P510S construct, a PCR reaction was used to amplifythe P510S coding region using MAPS-P510S-AW184 sense primers with EcoR Isites, and P510S-AW185 antisense primers for MAPS-P510S with Kpn I sitesand a stop codon. PCR product was purified from agarose gel, digestedwith EcoR I and Kpn I, and ligated to pCRX4 vector that had beenlinearized with the same two restriction enzymes. The ligation mixturewas transformed into E. coli and several clones were randomly selectedfor restriction enzyme analysis and DNA sequencing. MAPS-P510S/pCRX4 #1and #3 clones were confirmed. The determined DNA sequence for thisconstruct is set forth in SEQ ID NO: 1007, encoding the polypeptide ofSEQ ID NO: 1008.

Example 40 Detection of P501S and P703P-Specific Antibodies in Sera ofProstate Cancer Patients and Normal Donors

The identification and characterization of prostate specific antigensP501S and P703P are described above, illustrative forms of which are setforth in SEQ ID NO: 113 (encoded by SEQ ID NO: 110) and SEQ ID NO: 525(encoded by SEQ ID NO: 524), respectively. This example describes thedetection of antibodies specific for these proteins in the sera ofprostate cancer patients. Effective protein-based screening for prostateantigen-specific antibodies in patient sera has been problematic due toprotein instability and/or contamination of small amounts of E.coli-derived proteins. However, the carboxyamidation of recombinantP501S and P703P has provided improved stability for these proteins, asconfirmed by SDS-PAGE. Thus, carboxyamidated P501S and P703P proteinswere used to screen for P501S- and P703P-specific antibodies in the seraof 55 prostate cancer patients and 13 normal male donors, as follows.

Flat-bottomed immuno-plates were incubated and coated for at least 3hours at room temperature or overnight at 4 degrees C. with 50 ulcoating buffer (0.1N carbonate buffer; pH 9.5) supplemented with 600 ngof carboxyamidated P501S or 300 ng of carboxyamidated P703P protein. Thesolution was removed and at least 150 ul of blocking buffer (1:1 mixtureof 10% NFDM (Non Fat Dry Milk)/PBS and Superblock (Pierce) was added ineach well and incubated for 3 hours at room temp or overnight at 4degrees C. After washing at least 3 times with 0.05% Tween 20-containingPBS and once with PBS, 50 ul of sera from prostate cancer patients ornormal donors was diluted with 10% NGS (normal goat sera; Gibco BRL,#16210-072) containing PBS, added and incubated for 3 hours at roomtemperature or for overnight at 4 degrees C. After washing at least 3times with 0.05% Tween-containing PBS and once with PBS, 100 ul ofHRP-conjugated anti-human IgG (Jackson ImmunoResearch Laboratories Inc.;#709-036-149) (×8000 dilution by 1:1 mixture of 10% NFDM/PBS+Superblock)were added and incubated for 30-60 minutes at room temperature. Afterwashing at least 6 times with 0.05% Tween-containing PBS and once withPBS, 80-100 ul of the TMB-substrate solution was added and incubated atroom temperature. After obtaining suitable colors on the standard of IgGbetween 10-30 min, 40-50 ul (a half volume of substrate) of 0.1Nsulfuric acid solution was added to stop the enzyme reaction.Absorbances were then determined with a microplate reader (BiotecInstruments).

Using this approach, P501S-specific antibodies were detected in 32/55prostate cancer patients and 5/13 normal donors. P703-specificantibodies were detected in 24/55 prostate cancer patients and 0/13normal donors. These results confirm the in vivo immunogenicity of P501Sand P703P. Moreover, the results demonstrate that the carboxyamidatedforms of P501S and P703P retain the epitopes that are recognized byrabbit/mouse antibodies, as well as by antibodies from prostate cancerpatients.

Example 41 Generation of Monoclonal Antibodies to P711P

The identification and characterization of P711P (SEQ ID NO: 382,encoding the protein of SEQ ID NO: 383) is described above (e.g.,Example 3). For production and purification of P711P protein used forantibody generation, E. coli expressing recombinant P711P protein weregrown overnight in LB Broth with the appropriate antibiotics at 37° C.in a shaking incubator. The next morning, 10 ml of the overnight culturewas added to 500 ml of 2x YT plus appropriate antibiotics in a 2L-baffled Erlenmeyer flask. When the optical density (at 560 nanometers)of the culture reached 0.4-0.6, the cells were induced with IPTG (1 mM).Four hours after induction with IPTG the cells were harvested bycentrifugation. The cells were then washed with phosphate bufferedsaline and centrifuged again. The supernatant was discarded and thecells were either frozen for future use or immediately processed. Twentymilliliters of lysis buffer was added to the cell pellets and thesuspension was vortexed. To break open the E. coli cells, this mixturewas then run through a French Press at a pressure of 16,000 psi. Thecells were then centrifuged again and the supernatant and pellet werechecked by SDS-PAGE for the partitioning of the recombinant protein. Forproteins that localized to the cell pellet, the pellet was resuspendedin 10 mM Tris pH 8.0, 1% CHAPS and the inclusion body pellet was washedand centrifuged again. This procedure was repeated twice more. Thewashed inclusion body pellet was solubilized with either 8 M urea or 6 Mguanidine HCl containing 10 mM Tris pH 8.0 plus 10 mM imidazole. Thesolubilized protein was added to 5 ml of nickel-chelate resin (Qiagen)and incubated for 45 minutes to 1 hour at room temperature withcontinuous agitation. After incubation, the resin and protein mixturewere poured through a disposable column and the flow through wascollected. The column was then washed with 10-20 column volumes of thesolubilization buffer. The antigen was then eluted from the column using8M urea, 10 mM Tris pH 8.0 and 300 mM imidazole and collected in 3 mlfractions. A SDS-PAGE gel was run to determine which fractions to poolfor further purification.

As a final purification step, a strong anion exchange resin, e.g.,Hi-Prep Q (Biorad) was equilibrated with the appropriate buffer and thepooled fractions from above were loaded onto the column. Each antigenwas eluted off of the column with an increasing salt gradient. Fractionswere collected as the column was run and another SDS-PAGE gel was run todetermine which fractions from the column to pool. The pooled fractionswere dialyzed against 10 mM Tris pH 8.0. This material was thenevaluated for purity as determined by SDS-PAGE or HPLC, concentration asdetermined by Lowry assay or amino acid analysis, identity as determinedby amino terminal protein sequence, and endotoxin level was determinedby the Limulus (LAL) assay. The proteins were then vialed afterfiltration through a 0.22-micron filter and the antigens were frozenuntil needed for immunization.

To generate anti-P711P mouse monoclonal antibodies, mice were immunizedintraperitoneally (IP) with 50 micrograms of recombinant P711P proteinthat had been mixed to form an emulsion with an equal volume of CompleteFreund's Adjuvant (CFA). Every three weeks animals were injected i.p.with 50 micrograms of recombinant P711P protein that had been mixed withan equal volume of IFA to form an emulsion. After the fourth injection,spleens were isolated and standard hybridoma fusion procedures were usedto generate anti-P711P mouse monoclonal antibodies.

Anti-P711P monoclonal antibodies were screened by ELISA analysis usingthe bacterially expressed recombinant P711P protein. Forcharacterization of anti-P711P monoclonal antibodies by ELISA, 96 wellplates were coated with P711P antigen by incubating with 50 microliters(typically about 1 microgram) at 4 degrees C. for 20 hours. 250microliters of BSA blocking buffer was added to the wells and incubatedat room temperature for 2 hours. Plates were washed 6 times withPBS/0.01% TWEEN. Fifty microliters of each undiluted monoclonalsupernatant were added per well and incubated at room temperature for 30minutes. Plates were washed as described above before 50 microliters ofgoat anti-mouse horseradish peroxidase (HRP) at a 1:10000 dilution wasadded and incubated at room temperature for 30 minutes. Plates werewashed as described above and 100 μl of TMB Microwell PeroxidaseSubstrate was added to each well. Following a 15 minute incubation inthe dark at room temperature the colorimetric reaction was stopped with100 μl 1N H2SO4 and read immediately at 450 nm. For Western blotanalysis, recombinant P711P protein was diluted with SDS-PAGE loadingbuffer containing beta-mercaptoethanol, then boiled for 10 minutes priorto loading the SDS-PAGE gel. Protein was transferred to nitrocelluloseand probed with each of the anti-P711P hybridoma supernatants.Anti-mouse-HRP was used to visualize the anti-P711P reactive bands byincubation in ECL substrate. By this approach, at least seven anti-P711Pmonoclonal antibodies were generated and demonstrated to be reactivewith recombinant P711 protein by ELISA and/or Western analysis.

Example 42 Recognition of Carbamidomethylated CTL Epitope byP501S-Specific T-Cells

The identification and characterization of prostate specific antigenP501S has been described above, a representative full length DNAsequence for which is set forth in SEQ ID NO: 110, encoding the aminoacid sequence set forth in SEQ ID NO: 113. CTL specific for a peptide ofP501S (peptide 370-378, amino acid sequence CLSHSVAVV) were previouslydemonstrated to recognize naturally processed P501S as demonstrated byrecognition of P501S transduced target cells. In this example, mice wereimmunized with P501S DNA followed by a second immunization using aP501S-containing adenovirus delivery vector 14 days later. On day 35,spleen cells were harvested and stimulated in vitro weekly usingirradiated Jurkat A2 kb-P501 cells. CTL lines were assayed for lyticactivity against parental Jurkat A2Kb cells, Jurkat A2Kb cellstransduced with P501S, Jurkat A2Kb cells pulsed with p370-378, or JurkatA2KbT cells pulsed with the carboxy amidated p370-378 peptide. Usingthis approach, it was found that p370-378 specific T cells not onlyrecognize the synthetic unmodified 370-378 peptide, but also recognize acarbamidomethylated form of 370-378. These results are important fordemonstrating cross-recognition of naturally processed P501, anummodified synthetic peptide corresponding to amino acids 370-378,P501S, and the modified synthetic peptide 370-378. Thus,carbamidomethylation of recombinant P501S does not impair T cellrecognition of this CTL epitope.

Example 43 Preparation of P501S Fusion Proteins

The identification and characterization of prostate specific antigenP501S has been described above, a representative full length DNAsequence for which is set forth in SEQ ID NO: 110, encoding the aminoacid sequence set forth in SEQ ID NO: 113. In this example, illustrativeP501S fusion constructs were made and the encoded fusion proteinsproduced. Using sequence specific primers, the C-terminal truncatedregion of P501S (amino acids 341-527) was PCR amplified using thefollowing conditions:

10 μl 10×Pfu buffer

1 μl 10 mM dNTPs

2 μl 10 μM each oligo

83 μl sterile water

1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, Calif.)

50 ηg DNA

96° C. 2 minutes

96° C. 30 seconds 72° C. 1 minute 30 seconds×40 cycles

72° C. 4 minutes

The resulting PCR product was digested with NcoI and NotI and clonedinto the commercially available vector pET32 (Novagen, Madison Wis.)that was also digested with NcoI and NotI. The pET32 vector incorporatesan N-terminal fusion with the Trx antigen, which is Thioredoxin, an E.coli protein that can enhance expression for difficult to expressantigens, and can further enhance solubility of the resulting fusionprotein. The resulting cDNA sequence for the coding region of thisTrx-P501S fusion construct is set forth in SEQ ID NO: 1010, encoding thefusion amino acid sequence set forth in SEQ ID NO: 1011.

Example 44 Evaluation of P501S Immunogenicity

The identification and characterization of prostate specific antigenP501S has been described above, a representative full length DNAsequence for which is set forth in SEQ ID NO: 110, encoding the aminoacid sequence set forth in SEQ ID NO: 113. This example describes theresults of a preclinical in vivo immunogenicity study evaluatingvaccination with P501S protein (either unmodified or carboxy amidated),in combination with various adjuvants, compared with DNA/adenovirusP501S immunization. The following experimental design was employed:

Adjuvant Group Antigen System 1 10 ug P501S AS1 unmodified 2 10 ug P501SAS2 unmodified 3 10 ug P501S AS7 unmodified 4 10 ug P501S AS1carboxyamidated 5 10 ug P501S AS2 carboxyamidated 6 10 ug P501S AS7carboxyamidated 7 100 ug P501S None DNA/10⁸ pfu P501S adenovirus 8Buffer AS2. 9 Naïve — 3 immunizations, 2 weeks apart; Harvest 8 daysafter last immunization; Route = i.m. + f.p.; Mice = A2Tg females(5/group);

Assays were subsequently performed for evaluating CTL responses,antibody responses and CD4 responses (interferon-gamma & proliferation).The results of this evaluation demonstrated that both unmodified andcarboxy amidated P501S protein delivered in adjuvant are immunogenic, asassessed by antibody and CD4 T cell responses. The CD4 and antibodyresponses observed with protein administration were generally strongerthan the response observed with DNA/Adenovirus immunization. CTLresponses were not detected following protein immunization in theadjuvant systems tested. However, CTL were consistently detectablefollowing P501S adenovirus immunization. The antibody and CD4 responsesrecognized both unmodified P501S and P501S modified by carboxyamidation. Comparative evaluation of responses obtained with variousadjuvant systems demonstrated little effect on immunogenicity, with theexception that AS7 generated a stronger Th1 response.

Example 45 Identification of a Naturally Processed CTL Epitope for P501S

The identification and characterization of prostate specific antigenP501S has been described above, a representative full length DNAsequence for which is set forth in SEQ ID NO: 110, encoding the aminoacid sequence set forth in SEQ ID NO: 113. In this example, a naturallyprocessed, murine A2Kb CTL epitope has been identified for P501S. Thisepitope was mapped using a series of C-terminal deletion constructs,C-terminal subclones containing the last 92 amino acids of P501S, andsets of peptides spanning P501S regions of interest. The T cell clonesused to map the epitope were derived from limiting dilutions ofsplenocytes from P501S DNA immunized and P501S-adenovirus immunizedC57BL/6 A2Kb transgenic mice. Thirty-seven T cell clones were identifiedas reactive to P501S-transduced Jurkat A2Kb targets in CTL assays. Thelines, however, were not reactive to Jurkat A2Kb cells pulsed with apreviously identified naturally processed P501S epitope corresponding toamino acid residues 370-378 of SEQ ID NO: 113. This observation pointedto the existence of a second epitope other than the 9-mer epitope ofamino acid residues 370-378. The T cell clones were thus tested withJurkat A2Kb cells transduced with C-terminal deletion constructs denotedFragA-FragE. None of the T cell clones generated CTL responses to thesedeletion constructs, indicating that the epitope likely mapped withinthe C-terminal 92 amino acids of P501S (461-553). Six T cell clones weredemonstrated to be responsive to Jurkat A2Kb clones expressing P501Samino acid residues 462-553 or 462-517. Using a murine ELISPOT assay,these transduced targets, and Jurkat A2Kb targets pulsed with various20-mer, 15-mer, 10-mer and 9-mer peptides, allowed for theidentification of a naturally processed epitope for P501S, having thesequence SACDVSVRV, and corresponding to amino acid residues 464-472 ofSEQ ID NO: 113.

Example 46 Characterization of P790P Expression

The identification of prostate antigen P790P has been described above,the full length cDNA sequence for which is set forth in SEQ ID NO: 526,encoding the amino acid sequence set forth in SEQ ID NO: 527. Forrecombinant expression in mammalian cells, P790P was subcloned intopCEP4 and VR0113 (a polylinker modified VR1012 vector) mammalianexpression vectors. The constructs were transfected into HEK293 cells(ATCC) using Lipofectamine 2000 (Gibco). The HEK293 cells were plated ina 6 well dish at a density of 300,000 cells/ml in DMEM (Gibco)containing 10% FBS (Hyclone) and allowed to incubate for 6-8 hours. Oneug of DNA was then added to 100 ul of DMEM containing no FBS and 3 ul ofLipofectamine 2000 was added to 100 ul of DMEM containing no FBS. Bothwere incubated for 5 minutes at room temperature. The lipofectamine/DMEMmixture was added to the DNA/DMEM mixture and incubated for 15 minutesat room temperature. The lipofectamine 2000/DNA mix was then added tothe HEK293 cells and incubated for 48-72 hrs at 37 degrees C. with 7%CO2. Cells were collected and pelleted by centrifugation and washedtwice with PBS.

For Western blot analysis, whole cell lysates were generated by dilutingsamples directly into SDS-PAGE loading buffer containingbeta-mercaptoethanol, then incubated for 10 minutes at 70 degrees C.prior to loading the SDS-PAGE gel. Protein was transferred ontonitrocellulose and probed using the mouse anti-FLAG antibody (KodakScientific Imaging, Cat#IB13026) at a concentration of 10 ug/ml. Theblot was detected with goat anti-mouse-HRP followed by incubation in ECLsubstrate. An identical blot was probed with anti-P790 peptide sera(3-13-2001, 3875 L) at 1/1000. This blot was detected with goatanti-rabbit-HRP followed by incubation in ECL substrate. Using thisapproach, expression of recombinant P790P protein was detected intransiently transfected HEK293 cells using an anti-FLAG polyclonalantibody.

Example 47 Expression of P504S in Adenomatous Hyperplasia of theProstate

This example describes how the prostate antigen P504S, and antibodiesspecific for P504S, represent important diagnostic reagants useful inthe detection of prostate carcinoma, and, additionally, in thedifferentiation of atypical adenomatous hyperplasia (AAH) from prostatecarcinoma. AAH of the prostate, also known as adenosis, is characterizedby abnormal architectural patterns, but without significant cytologicatypia, in prostatic glandular epithelium. It is often difficult todistinguish AAH from prostatic carcinoma. In addition, it has beensuggested that AAH is a precursor lesion of the protatic adenocarcinoma.P504S (the DNA and amino acid sequences for which were disclosed in SEQID NOs: 107 and 108), is a protein that we have previously shown to behighly over-expressed in prostatice adenocarcinoma, and thereforerepresents an excellent marker for prostate cancer.

Here we use immunohistochemistry to determine the expression profile ofP504S in a number of different prostate samples. A total of 80 sampleswere examined, including 40 of AAH (prostatectomy: n=30, biopsy: n=6,and transurethral resection: n=4), 20 cases of prostatic adenocarcinomasand 20 cases of benign prostatic hyperplasia (BPH). Immunohistochemistryfor P504S and a basal cell specific marker, 34βE12, was performed on allsamples.

The 34βE12 stain confirmed the presence of patchy basal cells in all 40cases of AAH. P504S staining showed strong cytoplasmic granular stainingin 20/20 prostatic carcinomas and in 6 of 40 cases of AAH (15%). No BPHor benign prostate glands adjacent to carcinomas or adjacent to AAH werepositive for P504S. A diffuse staining pattern of P504S was found inprostate carcinoma samples, while focal positivity was seen in thepositive cases of AAH.

These findings demonstrate that combination staining using 34βE12 andP504S has a strong diagnostic value in distinguishing AAH from prostaticadenocarcinoma, and thus further validates the use of P504S in any of avariety of illustrative diagnostic and therapeutic embodiments describedherein, for example.

Example 48 Generation of an Illustrative P703P Fusion Protein

The identification and characterization of related forms of the prostatespecific antigen P703P has been described above, one representative DNAsequence for which is set forth in SEQ ID NO: 171, encoding an aminoacid sequence set forth in SEQ ID NO: 172. In this example, anillustrative P703P fusion construct was made and the encoded fusionprotein produced. This recombinant fusion protein can be used, forexample, in diagnostic methods for the detection of prostate cancerand/or to elicit an immune response in a subject for the preventionand/or treatment of prostate cancer.

Full length human PAP DNA was obtained by PCR from a prostate cancertissue cDNA library (PT library) with primers hPAPF1 and hPAPRV1. Thesequences for these primers are: hPAPF1

(SEQ ID NO: 1014) 5′CGGCGGATCCGCCGCCACCATGAGAGCTGCACCCCTCCTCCT-3′ andhPAPRV1 (SEQ ID NO: 1015)5′CGGCCTCGAGCTAATCTGTACTGTCTTCAGTACCTTGATGGCTG-3′.

The PCR product was then cloned into the PCR BluntII TOPO vector. Tomodify the hPAP a second PCR was performed with primers FOPP2F15′GAGAAAACCGTCCAGGCCAGTAAGGAGTTGAAGTTTGTGACTTTGGTG-3′ (SEQ ID NO: 1016)and HPAPRV1 (SEQ ID NO: 1015), using hPAP/TOPO BluntII as a template.

FOPP/PCRX1 (P703P containing DNA) was also modified using primers

FOPPF1 (SEQ ID NO: 1017)5′CGGCGGGCATATGCATCACCATCACCATCACATCATAAACGGCGAGGA CTGCAG-3′ andFOPP2RV1 (SEQ ID NO: 1018)5′CACCAAAGTCACAAACTTCAACTCCTTACTGGCCTGGACGGTTTT CTC-3′.

Finally, using primers FOPPF1 and hPAPRV1, and the modified hPAP andP703P PCR products as templates, a PCR was performed to generate FOPP2.This PCR product was then cloned in to the PCRX1 vector at therestriction enzyme cutting sites NdeI and XhoI. The FOPP2/PCRX1 plasmidwas then used to transform the E. coli strain Tuner (DE3) pLys S andplated out on LB plates containing kanamycin and chloamphenicol. UsingSDS-PAGE analysis and Western blot analysis with antibodies againstHis-Tag, the transformant was shown to express the FOPP2 protein whenexpression was induced using IPTG.

To generate large quantity of the FOPP2 protein, the expression cloneculture was inoculated into 20 ml of STB containing 50 μg/ml kanamycinand 34 μg/ml chloamphenicol, and grown at 37° C. until the OD600 readapproximately 1. The culture was then diluted into 1 liter of STBcontaining 50 μg/ml kanamycin and 34 μg/ml chloamphenicol, and grown at37° C. until the OD600 read approximately 0.4. IPTG was then added to afinal concentration of 1 mM, and the culture was incubated at 37° C. for2 hours. The cells were centrifuged at 5000 RPM for 8 minutes, washed inPBS, re-centrifuged, and stored at −70° C.

To purify the protein, the cell pellet was re-suspended in 25 ml of 10mM Tris-Cl (pH 8.0) containing 2 mM PMSF and protease inhibitors. Thecells were sonicated several times and the lysate was centrifuged for 60minutes at 4000 RPM. The precipitate, containing the inclusion bodies,was washed twice with 10 mM Tris-HCl (pH 8.0) containing 1% CHAPS. Theinclusion body was dissolved in 40 ml of binding buffer (10 mM Tris-HCl,pH 8.0, 100 mM sodium phosphate, and 8M urea). The solution was mixedwith 10 ml of Ni-NTA resin (Qiagen) for 30 minutes at room temperature.The mixture was then poured into a 25 ml column, and washed with 50 mlof wash buffer (10 mM Tris-Cl pH 6.3, 100 mM sodium phosphate, 0.5% DOCand 8M urea). The bound protein was then eluted using elution buffer (10mM Tris-Cl, pH 8.0, 350 mM Imidazole, 100 mM sodium phosphate, and 8Murea). The fractions containing FOPP2 were combined and dialyzedextensively using a large volume of Tris-Cl, pH 8.0, aliquoted, andstored at −70° C. The FOPP2 sequence was confirmed, and the DNA andamino acid sequence disclosed in SEQ ID NOs: 1019 and 1020,respectively.

Example 49 Expression of the Prostate Antigen P501S in E. Coli

This example describes the recombinant production of P501S polypeptidesthat are useful, for example, in immunodiagnostic and immunotherapeuticapplications. The identification and characterization of prostatespecific antigen P501S has been described above, a representative fulllength DNA sequence for which is set forth in SEQ ID NO: 110, encodingthe amino acid sequence set forth in SEQ ID NO: 113. This exampledescribes the expression of recombinant P501S C (corresponding to aminoacids 257-553) and P501S D (corresponding to amino acids 316-553)antigens using an E. coli expression system combined with an N-terminalhistadine tag and a TAT (twin arginine translocator) signal peptidefusion.

Expression of P501S C in E. coli:

The P501S C coding region was PCR amplified with the following primers:

(SEQ ID NO: 1023) PDM-930 5′cttccacggctgcaccagctgtgc3′ (SEQ ID NO: 1024)PDM-165 5′cacggacgtgaattctacgctgagtatttggcc3′Expression of P501S D in E. coli:

The P501S D coding region was PCR amplified with the following primers:

(SEQ ID NO: 1025) PDM-929 5′cactatgatgaaggcgttcggatgggcag3′ (SEQ ID NO:1024) PDM-165 5′cacggacgtgaattctacgctgagtatttggcc3′The PCR was performed with the following reaction components:

10 μl 10×Pfu buffer

1 μl 10 mM dNTPs

2 μl 10 μM of each primer

83 μl of sterile water

1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, Calif.)

50 ng DNA

PCR amplification was performed using the following reaction conditions:

96° C. for 2 minutes, followed by 40 cycles of:

96° C. for 20 seconds;

67° C. for 15 seconds; and

72° C. for 2 minutes, followed by a final extension step of:

72° C. for 4 minutes.

The PCR products were digested with EcoRI and cloned into pPDM and pTAT(pPDM using a TAT signal peptide leader) at the Eco721I and EcoRI.Constructs were confirmed to be correct through sequence analysis andtransformed into BLR (DE3) pLys S, HMS 174 (DE3) pLys S, Rosetta (DE3),and Rosetta (DE3) pLys S cells to confirm protein expression. The aminoacid sequence for the P501S C-His construct was confirmed, and isdisclosed in SEQ ID NO:1029, with the corresponding DNA sequencedisclosed in SEQ ID NO:1027. The amino acid sequence for the P501S D-Hisconstruct was confirmed, and is disclosed in SEQ ID NO:1028, with thecorresponding DNA sequence disclosed in SEQ ID NO:1026. Theserecombinant P501S polypeptides can be used, for example, inimmunodiagnostic and/or immunotherapeutic embodiments, such as thosedescribed herein.

Example 50 Expression of the Prostate Antigen P504S in MalignantNeoplasms

This example describes how the prostate antigen P504S, and antibodiesspecific for P504S, represent important therapeutic and diagnosticreagants useful in the detection of various types of carcinomas. P504S(the DNA and amino acid sequences for which were disclosed in SEQ IDNOs: 107 and 108), is a protein that we have previously shown to behighly over-expressed in prostatic adenocarcinoma, and thereforerepresents an excellent diagnostic marker for prostate cancer. Here,immunohistochemistry was used to determine the expression profile ofP504S in 515 cases of malignant neoplasms with epithelioid components.The tissue sections were stained with the anti-P504S antibody, 13H4, thegeneration of which was described in Example 18. Staining positive forP504S were 81% of hepatacellular carcinomas (17/21), 80% of colorectalcarcinomas (20/25), 75% of renal cell carcinomas (18/24), 31% ofurothelial carcinomas (9/29) and 27% of gastric carcinomas (4/15).Rarely staining positive for P504S were, lung, breast, pancreas, bileduct, adrenal gland, and salivary gland carcinomas. Consistentlynegative for P504S staining were ovary, thyroid and endometriumcarcinomas, melanomas, squamous cell carcinomas, basal cell carcinomas,soft tissue tumors including epitheloid sarcomas and synovial sarcoma,thmomas and germ cell tumors.

These findings identify additional tumor types demonstratingoverexpression of P504S and further validate the use of P504S in any ofa variety of illustrative diagnostic and therapeutic embodimentsdescribed herein.

Example 51 Extended cDNA Sequences for Prostate Antigens P704P, P712Pand P775P

This example describes additional cDNA sequences for the prostateantigens P704P, P712P, and P775P. Screening the partial sequencesspecific for the prostate antigens P704P, P712P, and P775P, against aprostate tumor library using colony hybridization and PCR, in additionto bioinformatics methods, led to the identification of additionalsequences for each of these antigens.

Sequences for prostate antigen P704P were previously provided in SEQ IDNOs:67, 402, and 699-701. Herein is provided an additional extended cDNAsequence for P704S, which is disclosed in SEQ ID NO:1036.

Sequences for prostate antigen P712P were previously provided in SEQ IDNOs:308, 397, and 552, with corresponding amino acid sequences disclosedin SEQ ID NOs:553-568. Herein is provided an additional extended cDNAsequence for P712S, which is disclosed in SEQ ID NO:1034.

Sequences for prostate antigen P775P were previously provided in SEQ IDNOs:311, 473-476, 553, and 593-597, with corresponding amino acidsequences disclosed in SEQ ID NOs:477-483 and 957-966. Herein isprovided an additional extended cDNA sequence for P712S, which isdisclosed in SEQ ID NO:1035.

In view of the previously identified over-expression of P704P, P712P,and P775P in prostate cancer, these additional extended sequences alsorepresent important reagants useful in the detection and/or therapy ofprostate cancer.

Example 52 The Identification of a P501S Minigene that Contains ThreeOverlapping Class I Epitopes for Multi-Epitope Vaccine Immunization

The identification and characterization of prostate specific antigenP501S has been described above, a representative full length DNAsequence for which is set forth in SEQ ID NO: 110, encoding the aminoacid sequence set forth in SEQ ID NO: 113. Extensive analysis of P501S(described in the above examples) including cDNA microarray,quantitative real-time PCR, and immunohistochemical analyses, have shownit to be expressed almost exclusively in normal prostate, benignprostate hyperplasia and both primary and secondary prostate tumors.

This example describes the identification of a small 11 amino acidfragment derived from P501S that contains naturally processed epitopesfor at least three class I alleles. The peptide corresponds to aminoacids 463-473 of P501S (SEQ ID NO:113), corresponding to the sequenceASACDVSVRVV (SEQ ID NO:1037, with the corresponding polynucleotidesequence disclosed in SEQ ID N:1038). This sequence was derived from theprevious identification of 6 different CD8+ T cell epitopes. Example 9described the identification of two HLA-Cw*0501 restricted epitopes,SACDVSVRVV (SEQ ID NO:978) and ACDVSVRVV (SEQ ID NO:1012). Example 12described the identification of two HLA-B*51011 restricted epitopes,ASACDVSVRV (SEQ ID NO:855) and SACDVSVRV (SEQ ID NO:858). Example 48described the identification of two HLA-A*0201 restricted epitopes,ASACDVSVRV and SACDVSVRV.

To examine the potential cross-reactivity of the HLA-B*51011 andHLA-Cw*0501 epitopes identified in the human in-vitro system, d310B-LCL, which endogenously express both the HLA-B*51011 and HLA-Cw*0501alleles, were pulsed with each of the purified peptides SACDVSVRV,SACDVSVRVV, and ACDVSVRVV, and used as APC for the HLA-B*51011restricted clone 2A2-4E5 and the HLA-Cw*0501 restricted clone 2H2-1A12.The 2A2-4E5 clone specifically recognized the SACDVSVRV peptide, andfailed to recognize either the SACDVSVRVV or ACDVSVRVV peptides.Conversely, and in agreement with the previously described finespecificity of this clone, the 2H2-1A12 clone specifically recognizedboth the SACDVSVRVV and ACDVSVRVV peptides. However, 2H2-1A12 failed torecognize the SACDVSVRV peptide. Thus the two clones show nocross-reactivity of peptide recognition. Further evidence for theabsence of cross-reactivity between the two clones at the level of theMHC:peptide is the fact that each of the two clones failed to recognizeP501S expressing cells that express the reciprocal restricting allele.Example 45 describes the identification of an HLA-A2 restricted epitopederived from P501S using HLA-A2 kb transgenic mice. This epitopecomprises residues 464-475 of P501S.

Thus, 3 distinct class-I restricted epitopes are processed from a 11amino acid region of P501S. The phenomenon of a single T cell epitopebeing presented to T cells by more than one HLA allele has beenpreviously described. Specifically, a single MAGE-AL-derived epitope wasshown to be presented to T cells by both HLA-B*35 and HLA-A*1 alleles(Luiten et al, Tissue Antigens 56: 77-81, 2000). Although B and T cellepitope clustering has been previously reported (Bellone et al, 1995),to our knowledge this is the first demonstration of multiple T cellepitopes being identified to be clustered to a minimal amino acidsequence. The observation that at least one of the 3 epitopes appears tobe processed less efficiently suggests that the clustering of theepitopes is not simply a function of preferential processing of thisregion. The clustering of multiple T cell epitopes to this small aminoacid sequence indicates that this region of P501S will be particularlyuseful in the development of P501S-specific T cell vaccinationstrategies.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. An isolated polypeptide comprising no more than X amino acid residuesof SEQ ID NO: 113, wherein said polypeptide comprises SEQ ID NO:1037,and wherein X is selected from the group consisting of 11-542.
 2. Thepolypeptide of claim 1, wherein SEQ ID NO: 1037 comprises an 11 aminoacid fragment of SEQ ID NO: 113 that contains naturally processed T-cellepitopes for three Class I MHC alleles.
 3. The polypeptide of claim 1,wherein X is selected from the group consisting of 11-100.
 4. Thepolypeptide of claim 1, wherein X is selected from the group consistingof 11-50.
 5. The polypeptide of claim 1, wherein X is selected from thegroup consisting of 11-25.
 6. An isolated polynucleotide comprising asequence selected from the group consisting of: (a) sequences providedin SEQ ID NOs:1034-1036; (b) complements of the sequences provided inSEQ ID NOs: 1034-1036; (c) sequences consisting of at least 20contiguous residues of a sequence provided in SEQ ID NOs: 1034-1036; (d)sequences that hybridize to a sequence provided in SEQ ID NOs:1034-1036, under highly stringent conditions; (e) sequences having atleast 75% identity to a sequence of SEQ ID NOs: 1034-1036; (f) sequenceshaving at least 90% identity to a sequence of SEQ ID NOs: 1034-1036; and(g) degenerate variants of a sequence provided in SEQ ID NOs: 1034-1036;and (h) sequences encoding a polypeptide according to claim
 1. 7. Anisolated polypeptide comprising an amino acid sequence selected from thegroup consisting of: (a) sequences encoded by a polynucleotide of claim6; and (b) sequences having at least 70% identity to a sequence encodedby a polynucleotide of claim 6; (c) sequences having at least 90%identity to a sequence encoded by a polynucleotide of claim 6; (d)sequences consisting of at least 5 contiguous amino acids of a sequenceencoded by a polynucleotide of claim 6; (e) sequences consisting of atleast 10 contiguous amino acids of a sequence encoded by apolynucleotide of claim 6; and (f) sequences consisting of at least 20contiguous amino acids of a sequence encoded by a polynucleotide ofclaim
 6. 8. An expression vector comprising a polynucleotide of claim 6operably linked to an expression control sequence.
 9. A host celltransformed or transfected with an expression vector according to claim8.
 10. An isolated antibody, or antigen-binding fragment thereof, thatspecifically binds to a polypeptide of claims 1 or
 7. 11. A method fordetecting the presence of a cancer in a patient, comprising the stepsof: (a) obtaining a biological sample from the patient; (b) contactingthe biological sample with a binding agent that binds to a polypeptideof claims 1 or 7 or a polypeptide of SEQ ID NO: 108; (c) detecting inthe sample an amount of polypeptide that binds to the binding agent; and(d) comparing the amount of polypeptide to a predetermined cut-off valueand therefrom determining the presence of a cancer in the patient.
 12. Afusion protein comprising at least one polypeptide according to claims 1or
 7. 13. An oligonucleotide that hybridizes to a sequence recited inSEQ ID NOs: 1034-1036 under highly stringent conditions.
 14. A methodfor stimulating and/or expanding T cells specific for a tumor protein,comprising contacting T cells with at least one component selected fromthe group consisting of: (a) polypeptides according to claims 1 or 7;(b) polynucleotides according to claim 6; and (c) antigen-presentingcells that express a polynucleotide according to claim 6, underconditions and for a time sufficient to permit the stimulation and/orexpansion of T cells.
 15. An isolated T cell population, comprising Tcells prepared according to the method of claim
 14. 16. A compositioncomprising a first component selected from the group consisting ofphysiologically acceptable carriers and immunostimulants, and a secondcomponent selected from the group consisting of: (a) polypeptidesaccording to claims 1 or 7; (b) polynucleotides according to claim 6;(c) antibodies according to claim 10; (d) fusion proteins according toclaim 12; (e) T cell populations according to claim 15; and (f) antigenpresenting cells that express a polypeptide according to claims 1 or 7.17. A method for stimulating an immune response in a patient, comprisingadministering to the patient a composition of claim
 16. 18. A method forthe treatment of a prostate cancer in a patient, comprisingadministering to the patient a composition of claim
 16. 19. A method fordetermining the presence of a cancer in a patient, comprising the stepsof: (a) obtaining a biological sample from the patient; (b) contactingthe biological sample with an oligonucleotide according to claim 13 oran oligonucleotide specific for SEQ ID NO: 107; (c) detecting in thesample an amount of a polynucleotide that hybridizes to theoligonucleotide; and (d) comparing the amount of polynucleotide thathybridizes to the oligonucleotide to a predetermined cut-off value, andtherefrom determining the presence of the cancer in the patient.
 20. Adiagnostic kit comprising at least one oligonucleotide according toclaim
 13. 21. A diagnostic kit comprising at least one antibodyaccording to claim 10 and a detection reagent, wherein the detectionreagent comprises a reporter group.
 22. A method for the treatment ofprostate cancer in a patient, comprising the steps of: (a) incubatingCD4+ and/or CD8+ T cells isolated from a patient with at least onecomponent selected from the group consisting of: (i) polypeptidesaccording to claims 1 or 7; (ii) polynucleotides according to claim 6;and (iii) antigen presenting cells that express a polypeptide of claims1 or 7, such that T cell proliferate; (b) administering to the patientan effective amount of the proliferated T cells, and thereby inhibitingthe development of a cancer in the patient.